Carbonate containing lipid compounds and compositions for intracellular delivery of therapeutic agents

ABSTRACT

The disclosure features novel lipids and compositions involving the same. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) further including therapeutic and/or prophylactics such as RNA are useful in the delivery of therapeutic and/or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Application No. 62/902,929, filed Sep. 19, 2019, the entirecontent of which is incorporated herein by reference.

FIELD OF DISCLOSURE

The present disclosure provides novel compounds, compositions comprisingsuch compounds, and methods involving lipid nanoparticle compositions todeliver one or more therapeutic and/or prophylactics to and/or producepolypeptides in mammalian cells or organs. In addition to a novel lipid,lipid nanoparticle compositions of the disclosure may include one ormore cationic and/or ionizable amino lipids, phospholipids includingpolyunsaturated lipids, PEG lipids, structural lipids, and/ortherapeutic and/or prophylactics in specific fractions.

BACKGROUND OF THE DISCLOSURE

The effective targeted delivery of biologically active substances suchas small molecule drugs, proteins, and nucleic acids represents acontinuing medical challenge. In particular, the delivery of nucleicacids to cells is made difficult by the relative instability and lowcell permeability of such species. Thus, there exists a need to developmethods and compositions to facilitate the delivery of therapeuticand/or prophylactics such as nucleic acids to cells.

Lipid-containing nanoparticle compositions, liposomes, and lipoplexeshave proven effective as transport vehicles into cells and/orintracellular compartments for biologically active substances such assmall molecule drugs, proteins, and nucleic acids. Such compositionsgenerally include one or more “cationic” and/or amino (ionizable)lipids, phospholipids including polyunsaturated lipids, structurallipids (e.g., sterols), and/or lipids containing polyethylene glycol(PEG lipids). Cationic and/or ionizable lipids include, for example,amine-containing lipids that can be readily protonated. Though a varietyof such lipid-containing nanoparticle compositions have beendemonstrated, improvements in safety, efficacy, and specificity arestill lacking.

SUMMARY OF THE DISCLOSURE

The present disclosure provides novel compounds and compositions andmethods involving the same.

In some aspects, the present disclosure relates to compound of Formula(A):

or its N-oxide, or a salt or isomer thereof, wherein

m is selected from 5, 6, 7, 8, and 9;

R² and R³ are each independently selected from the group consisting ofH, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

M and M′ are each independently selected from —OC(O)O— and —C(O)O—;

wherein at least one of M and M′ is —OC(O)O—;

R′^(a) is C₁₋₁₈ alkyl or C₂₋₁₈ alkenyl; and

R″ is a C₃-C₁₃ alkyl, optionally substituted with OH.

DETAILED DESCRIPTION

The disclosure relates to novel lipids and lipid nanoparticles (e.g.,empty LNPs or loaded LNPs) including a novel lipid. The disclosure alsoprovides methods of delivering a therapeutic and/or prophylactic to amammalian cell, specifically delivering a therapeutic and/orprophylactic to a mammalian organ, producing a polypeptide of interestin a mammalian cell, improving levels of protein produced in a mammaliancell as compared to LNPs comprising other lipids, and treating a diseaseor disorder in a mammal in need thereof. For example, a method ofproducing a polypeptide of interest in a cell involves contacting ananoparticle comprising an mRNA with a mammalian cell, whereby the mRNAmay be translated to produce the polypeptide of interest. A method ofdelivering a therapeutic and/or prophylactic to a mammalian cell ororgan may involve administration of a nanoparticle composition includingthe therapeutic and/or prophylactic to a subject, in which theadministration involves contacting the cell or organ with thecomposition, whereby the therapeutic and/or prophylactic is delivered tothe cell or organ. Such methods of delivery can be in vitro or in vivo.

The present disclosure provides lipids including a central amine moietyand at least one biodegradable group. The lipids described herein may beadvantageously used in lipid nanoparticle compositions for the deliveryof therapeutic and/or prophylactics to mammalian cells or organs. Forexample, the lipids described herein have little or no immunogenicity.For example, the lipid compound of Formula (A), (A1), (A2), (A3), (B),(I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb) has a lower immunogenicity as comparedto a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, aformulation comprising a lipid disclosed herein and a therapeutic orprophylactic agent has an increased therapeutic index as compared to acorresponding formulation which comprise a reference lipid (e.g., MC3,KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

In some aspects, the disclosure relates to a compound of Formula (B):

wherein

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

R^(aα), R^(aβ), R^(aγ) are each independently selected from the groupconsisting of H, C₁₋₁₂ alkyl, and C₂₋₁₂ alkenyl, wherein at least one ofR^(aα), R^(aβ), R^(aγ) is selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl; and

R′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl.

In some aspects, the disclosure relates to a compound of Formula (A1),(A2), or (A3):

wherein

m is selected from 5, 6, 7, 8, and 9;

R² and R³ are each independently selected from the group consisting ofH, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

R′^(a) is:

R^(aα), R^(aβ), and R^(aγ) are each independently selected from thegroup consisting of H, C₁₋₁₂ alkyl, and C₂₋₁₂ alkenyl, wherein at leastone of R^(aα), R^(aβ), and R^(aγ) is selected from the group consistingof C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;

R″ is a C₃-C₁₃ alkyl, optionally substituted with OH; and

R′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl.

In some aspects, the disclosure relates to a compound of Formula (I):

or its N-oxide, or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,−R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*″, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M is selected from —OC(O)O—,—OC(O)—NRM—C(O)O—, —O-M″—O—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,SO—, —OS—, —S(R^(M))₂O—, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, in which M″ isa bond,—(CH₂)_(z)C(O)—, C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—,—S(R**)₂—, or —S(O)—, wherein z is 1, 2, 3, or 4;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O—, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkyl-aryl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*′, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some aspects, the disclosure relates to a compound of Formula (I′):

or its N-oxide,

or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*″, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl, C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

M′ is selected from —OC(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,SO—, —OS—, —S(R^(M))₂O—, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, in which M″ isa bond,—(CH₂)_(z)C(O)—, C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—,—S(R**)₂—, or —S(O)—, wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle;

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some aspects, the disclosure relates to a compound of Formula (I-X):

or its N-oxide, or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*″, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In certain embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (IA):

or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; and R² and R³are independently selected from the group consisting of H, C₁₋₁₄ alkyl,and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (II):

or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from1, 2, 3, 4, and 5; and R² and R³ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In other embodiments, a subset of compounds of Formula (II) includesthose of Formula (IIa):

or its N-oxide, or a salt or isomer thereof, wherein

1 is selected from 1, 2, 3, 4, and 5;

M₁ is M′; and

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In other embodiments, a subset of compounds of Formula (II) includesthose of Formula (IIb):

or its N-oxide, or a salt or isomer thereof, wherein

1 is selected from 1, 2, 3, 4, and 5;

M₁ is M′; and

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Ia):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Ib):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Ic):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Id):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Ie):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (If):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In other embodiments, a subset of compounds of Formula (I) or (I′)includes those of Formula (Ig):

or its N-oxide, or a salt or isomer thereof, wherein R″ is a linear orbranched C₃-C₁₃ alkyl, optionally substituted with OH, and m is selectedfrom 5, 6, 7, 8, and 9.

In some aspects, the disclosure relates to a compound of Formula (I-Y):

or its N-oxide, or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*″, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O—, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*′, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I-Y) includesthose of Formula (I-Ya):

or its N-oxide, or a salt or isomer thereof, wherein each R″independently is selected from H, OH, and C₁₋₆ alkyl, 1 is selected from2, 3, 4, 5, and 6, and m is selected from 5, 6, 7, 8, and 9.

The compounds of any one of Formula (A), (A1), (A2), (A3), (B), (I),(I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If),(Ig), (II), (IIa), or (IIb) include one or more of the followingfeatures when applicable.

In some embodiments, R′a is :

wherein

denotes a point of attachment;

R^(aα), R^(aβ), and R^(aγ) are each independently selected from thegroup consisting of H, C₁₋₁₂ alkyl, and C₂₋₁₂ alkenyl, wherein at leastone of R^(aα), R^(aβ), and R^(aγ) is selected from the group consistingof C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; and

R′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl.

In some embodiments, M and M′ are each —OC(O)O—. In some embodiments, Mis

—C(O)O— and M′ is —OC(O)O—. In some embodiments, M is —OC(O)O— and M′ is—C(O)O—.

In some embodiments, 1 is 1, 3, or 5. In some embodiments, 1 is 1, 3, or4. In some embodiments, 1 is 5.

In some embodiments, R⁴ is

In some embodiments, R⁴ is —(CH₂)₂OH.

In some embodiments, R² and R³ are independently C₃₋₁₄ alkyl or C₃₋₁₄alkenyl.

In some embodiments, R′^(a) is selected from C₄ alkyl, C₄ alkenyl, C₅alkyl, C₅ alkenyl, C₆ alkyl, C₆ alkenyl, C₇ alkyl, C₇ alkenyl, C₉ alkyl,C₉ alkenyl, C₁₁ alkyl, C₁₁ alkenyl, C₁₇ alkyl, C₁₇ alkenyl, C₁₈ alkyl,and C₁₈ alkenyl, each of which is either linear or branched.

In some embodiments, R′ is C₄ alkyl or C₄ alkenyl. In some embodiments,R′ is C₅ alkyl or C₅ alkenyl. In some embodiments, R′ is C₆ alkyl or C₆alkenyl. In some embodiments, R′ is C₇ alkyl or C₇ alkenyl. In someembodiments, R′ is C₅ alkyl or C₈ alkenyl. In some embodiments, R′ is C₉alkylor C₉ alkenyl. In some embodiments, R′ is C₁₀ alkyl or C₁₀ alkenyl.In some embodiments, R′ is C₁₁ alkyl or C₁₁ alkenyl.

In certain embodiments, R′^(a) is branched C₁₋₁₈ alkyl. For example,R′^(a) is

In some embodiments, R″ is selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl. In some embodiments, R″ is C₃ alkyl, C₄ alkyl,C₅ alkyl, C₆ alkyl, C₇ alkyl, or C₈ alkyl. In some embodiments, R″ is C₉alkyl, C₁₀ alkyl, C₁₁ alkyl, C12 alkyl, C₁₃ alkyl, C₁₄ alkyl, or C₁₅alkyl.

In some embodiments, R² and R³ are independently C₅₋₁₄ alkyl or C₅₋₁₄alkenyl.

In some embodiments, R² and R³ are the same. In some embodiments, R² andR³ are C₈ alkyl. In certain embodiments, R² and R³ are C₂ alkyl. Inother embodiments, R² and R³ are C₃ alkyl. In some embodiments, R² andR³ are C₄ alkyl. In certain embodiments, R² and R³ are C₅ alkyl. Inother embodiments, R² and R³ are C₆ alkyl. In some embodiments, R² andR³ are C₇ alkyl.

In other embodiments, R² and R³ are different. In certain embodiments,R² is C₈ alkyl. In some embodiments, R³ is C₁₋₇ (e.g., C₁, C₂, C₃, C₄,C₅, C₆, or C₇ alkyl) or C₉ alkyl.

In some embodiments, R³ is C₁ alkyl. In some embodiments, R³ is C₂alkyl. In some embodiments, R³ is C₃ alkyl. In some embodiments, R³ isC₄ alkyl. In some embodiments, R³ is C₅ alkyl. In some embodiments, R³is C₆ alkyl. In some embodiments, R³ is C₇ alkyl. In some embodiments,R³ is C₉ alkyl.

In some embodiments, m is 5, 6, 7, 8, or 9. In some embodiments, m is 5,7, or 9. For example, in some embodiments, m is 5. For example, in someembodiments, m is 7. For example, in some embodiments, m is 9.

In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. Insome embodiments, n is 4. In some embodiments, n is not 3.

In some embodiments, n2 is 2, 3, or 4. In some embodiments, n2 is 2. Insome embodiments, n2 is 4. In some embodiments, n2 is not 3.

In some embodiments, the compound of Formula (A), (A1), (A2), (A3), (B),(I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb)is selected from Table 1.

TABLE 1 Amino Lipids  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

The central amine moiety of a lipid according to Formula (A), (A1),(A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic),(Id), (Ie), (If), (Ig), (II), (IIa), or (IIb) may be protonated at aphysiological pH. Thus, a lipid may have a positive or partial positivecharge at physiological pH. Such lipids may be referred to as cationicor ionizable (amino)lipids. Lipids may also be zwitterionic, i.e.,neutral molecules having both a positive and a negative charge.

Definitions

As used herein, the term “alkyl” or “alkyl group” means a linear orbranched, saturated hydrocarbon including one or more carbon atoms(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms), which is optionallysubstituted. The notation “C₁₋₁₄ alkyl” means an optionally substitutedlinear or branched, saturated hydrocarbon including 1-14 carbon atoms.Unless otherwise specified, an alkyl group described herein refers toboth unsubstituted and substituted alkyl groups.

As used herein, the term “alkenyl” or “alkenyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one double bond, which isoptionally substituted. The notation “C₂₋₁₄ alkenyl” means an optionallysubstituted linear or branched hydrocarbon including 2-14 carbon atomsand at least one carbon-carbon double bond. An alkenyl group may includeone, two, three, four, or more carbon-carbon double bonds. For example,C₁₈ alkenyl may include one or more double bonds. A C₁₈ alkenyl groupincluding two double bonds may be a linoleyl group. Unless otherwisespecified, an alkenyl group described herein refers to bothunsubstituted and substituted alkenyl groups.

As used herein, the term “alkynyl” or “alkynyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one carbon-carbon triplebond, which is optionally substituted. The notation “C₂₋₁₄ alkynyl”means an optionally substituted linear or branched hydrocarbon including2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynylgroup may include one, two, three, four, or more carbon-carbon triplebonds. For example, C₁₈ alkynyl may include one or more carbon-carbontriple bonds.

Unless otherwise specified, an alkynyl group described herein refers toboth unsubstituted and substituted alkynyl groups.

As used herein, the term “carbocycle” or “carbocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings of carbon atoms. Rings may be three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, or twenty membered rings. The notation“C₃₋₆ carbocycle” means a carbocycle including a single ring having 3-6carbon atoms. Carbocycles may include one or more carbon-carbon doubleor triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl oraryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl,cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term“cycloalkyl” as used herein means a non-aromatic carbocycle and may ormay not include any double or triple bond. Unless otherwise specified,carbocycles described herein refers to both unsubstituted andsubstituted carbocycle groups, i.e., optionally substituted carbocycles.In some embodiments, the carbocycle is a C₃₋₈ cycloalkyl. In someembodiments, the carbocycle is a C₃₋₆ cycloalkyl. In some embodiments,the carbocycle is a C₆₋₁₀ aryl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc. In some embodiments, an “aryl” is aC₆₋₁₀ carbocycle with aromatity (e.g. an “aryl” is a C₆₋₁₀ aryl).

As used herein, the term “heterocycle” or “heterocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings, where at least one ring includes at least one heteroatom.Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms.Rings may be three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, or fourteen membered rings. Heterocycles may includeone or more double or triple bonds and may be non-aromatic or aromatic(e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocyclesinclude imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl,thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl,isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl,furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl,and isoquinolyl groups. The term “heterocycloalkyl” as used herein meansa non-aromatic heterocycle and may or may not include any double ortriple bond. Unless otherwise specified, heterocycles described hereinrefers to both unsubstituted and substituted heterocycle groups, i.e.,optionally substituted heterocycles. In some embodiments, theheterocycle is a 4 to 12-membered heterocycloalkyl. In some embodiments,the heterocycle is a 5- or 6-membered heteroaryl.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6- , or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen sulfur, andboron. The nitrogen atom may be substituted or unsubstituted (i.e., N orNR wherein R is H or other substituents, as defined). The nitrogen andsulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p),where p=1 or 2). It is to be noted that total number of S and O atoms inthe aromatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, quinoline, isoquinoline, naphthrydine, indole,benzofuran, purine, benzofuran, deazapurine, indolizine.

As used herein, a “biodegradable group” is a group that may facilitatefaster metabolism of a lipid in a mammalian entity. A biodegradablegroup may be selected from the group consisting of, but is not limitedto, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—,—SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and aheteroaryl group. As used herein, an “aryl group” is an optionallysubstituted carbocyclic group including one or more aromatic rings.Examples of aryl groups include phenyl and naphthyl groups. As usedherein, a “heteroaryl group” is an optionally substituted heterocyclicgroup including one or more aromatic rings. Examples of heteroarylgroups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, andthiazolyl. Both aryl and heteroaryl groups may be optionallysubstituted. For example, M and M′ can be selected from the non-limitinggroup consisting of optionally substituted phenyl, oxazole, andthiazole. In the formulas herein, M and M′ can be independently selectedfrom the list of biodegradable groups above. Unless otherwise specified,aryl or heteroaryl groups described herein refers to both unsubstitutedand substituted groups, i.e., optionally substituted aryl or heteroarylgroups.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupsmay be optionally substituted unless otherwise specified. Optionalsubstituents may be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g.,

—OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxy groupsthat can be the same or different and R″″ is an alkyl or alkenyl group),a phosphate (e.g., P(O)₄ ³⁻), a thiol (e.g., —SH), a sulfoxide (e.g.,—S(O)R), a sulfinic acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)₂OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)₄ ²⁻), asulfonyl (e.g., —S(O)₂—), an amide (e.g., —C(O)NR₂, or—N(R)C(O)R), an azido (e.g., -N3), a nitro (e.g.,—NO2), a cyano(e.g.,—CN), an isocyano (e.g., —NC), an acyloxy (e.g.,—OC(O)R), an amino(e.g., —NR₂, —NRH, or —NH₂), a carbamoyl (e.g., —OC(O)NR₂, —OC(O)NRH, or—OC(O)NH₂), a sulfonamide (e.g., —S(O)₂NR₂, —S(O)₂NRH, —S(O)₂NH2,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)S(O)₂H, or —N(H)S(O)₂H), an alkyl group,an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl)group. In any of the preceding, R is an alkyl or alkenyl group, asdefined herein. In some embodiments, the substituent groups themselvesmay be further substituted with, for example, one, two, three, four,five, or six substituents as defined herein. For example, a C₁₋₆ alkylgroup may be further substituted with one, two, three, four, five, orsix substituents as described herein.

Compounds of the disclosure that contain nitrogens can be converted toN-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the disclosure. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the disclosure can beconverted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxycompounds can be prepared by oxidation of the parent amine by anoxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

About, Approximately: As used herein, the terms “approximately” and“about,” as applied to one or more values of interest, refer to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value). For example, when used in the contextof an amount of a given compound in a lipid component of a nanoparticlecomposition, “about” may mean +/−10% of the recited value. For instance,a nanoparticle composition including a lipid component having about 40%of a given compound may include 30-50% of the compound.

As used herein, the term “compound,” is meant to include all isomers andisotopes of the structure depicted. “Isotopes” refers to atoms havingthe same atomic number but different mass numbers resulting from adifferent number of neutrons in the nuclei. For example, isotopes ofhydrogen include tritium and deuterium. Further, a compound, salt, orcomplex of the present disclosure can be prepared in combination withsolvent or water molecules to form solvates and hydrates by routinemethods.

As used herein, the term “contacting” means establishing a physicalconnection between two or more entities. For example, contacting amammalian cell with a nanoparticle composition means that the mammaliancell and a nanoparticle are made to share a physical connection. Methodsof contacting cells with external entities both in vivo and ex vivo arewell known in the biological arts. For example, contacting ananoparticle composition and a mammalian cell disposed within a mammalmay be performed by varied routes of administration (e.g., intravenous,intramuscular, intradermal, and subcutaneous) and may involve variedamounts of lipid nanoparticles (e.g., empty LNPs or loaded LNPs).Moreover, more than one mammalian cell may be contacted by ananoparticle composition.

As used herein, the term “delivering” means providing an entity to adestination. For example, delivering a therapeutic and/or prophylacticto a subject may involve administering a nanoparticle compositionincluding the therapeutic and/or prophylactic to the subject (e.g., byan intravenous, intramuscular, intradermal, or subcutaneous route).

Administration of a nanoparticle composition to a mammal or mammaliancell may involve contacting one or more cells with the nanoparticlecomposition.

As used herein, the term “enhanced delivery” means delivery of more(e.g., at least 1.5 fold more, at least 2-fold more, at least 3-foldmore, at least 4-fold more, at least 5-fold more, at least 6-fold more,at least 7-fold more, at least 8-fold more, at least 9-fold more, atleast 10-fold more) of a therapeutic and/or prophylactic by ananoparticle to a target tissue of interest (e.g., mammalian liver)compared to the level of delivery of a therapeutic and/or prophylacticby a control nanoparticle to a target tissue of interest (e.g., MC3,KC2, or DLinDMA). The level of delivery of a nanoparticle to aparticular tissue may be measured by comparing the amount of proteinproduced in a tissue to the weight of said tissue, comparing the amountof therapeutic and/or prophylactic in a tissue to the weight of saidtissue, comparing the amount of protein produced in a tissue to theamount of total protein in said tissue, or comparing the amount oftherapeutic and/or prophylactic in a tissue to the amount of totaltherapeutic and/or prophylactic in said tissue. It will be understoodthat the enhanced delivery of a nanoparticle to a target tissue need notbe determined in a subject being treated, it may be determined in asurrogate such as an animal model (e.g., a rat model). In certainembodiments, a nanoparticle composition including a compound accordingto

Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (fib)has substantively the same level of delivery enhancement regardless ofadministration routes. For example, certain compounds disclosed hereinexhibit similar delivery enhancement when they are used for delivering atherapeutic and/or prophylactic either intravenously or intramuscularly.In other embodiments, certain compounds disclosed herein exhibit ahigher level of delivery enhancement when they are used for delivering atherapeutic and/or prophylactic intramuscularly than intravenously.

As used herein, the term “specific delivery,” “specifically deliver,” or“specifically delivering” means delivery of more (e.g., at least 1.5fold more, at least 2-fold more, at least 3-fold more, at least 4-foldmore, at least 5-fold more, at least 6-fold more, at least 7-fold more,at least 8-fold more, at least 9-fold more, at least 10-fold more) of atherapeutic and/or prophylactic by a nanoparticle to a target tissue ofinterest (e.g., mammalian liver) compared to an off-target tissue (e.g.,mammalian spleen). The level of delivery of a nanoparticle to aparticular tissue may be measured by comparing the amount of proteinproduced in a tissue to the weight of said tissue, comparing the amountof therapeutic and/or prophylactic in a tissue to the weight of saidtissue, comparing the amount of protein produced in a tissue to theamount of total protein in said tissue, or comparing the amount oftherapeutic and/or prophylactic in a tissue to the amount of totaltherapeutic and/or prophylactic in said tissue. For example, forrenovascular targeting, a therapeutic and/or prophylactic isspecifically provided to a mammalian kidney as compared to the liver andspleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold moretherapeutic and/or prophylactic per 1 g of tissue is delivered to akidney compared to that delivered to the liver or spleen followingsystemic administration of the therapeutic and/or prophylactic. It willbe understood that the ability of a nanoparticle to specifically deliverto a target tissue need not be determined in a subject being treated, itmay be determined in a surrogate such as an animal model (e.g., a ratmodel).

As used herein, “encapsulation efficiency” refers to the amount of atherapeutic and/or prophylactic that becomes part of a nanoparticlecomposition, relative to the initial total amount of therapeutic and/orprophylactic used in the preparation of a nanoparticle composition. Forexample, if 97 mg of therapeutic and/or prophylactic are encapsulated ina nanoparticle composition out of a total 100 mg of therapeutic and/orprophylactic initially provided to the composition, the encapsulationefficiency may be given as 97%. As used herein, “encapsulation” mayrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

As used herein, “encapsulation”, “encapsulated”, “loaded”, and“associated” may refer to complete, substantial, or partial enclosure,confinement, surrounding, or encasement. As used herein, “encapsulation”or “association” may refer to the process of confining an individualnucleic acid molecule within a nanoparticle and/or establishing aphysiochemical relationship between an individual nucleic acid moleculeand a nanoparticle. As used herein, an “empty nanoparticle” may refer toa nanoparticle that is substantially free of a therapeutic orprophylactic agent. As used herein, an “empty nanoparticle” or an “emptylipid nanoparticle” may refer to a nanoparticle that is substantiallyfree of a nucleic acid. As used herein, an “empty nanoparticle” or an“empty lipid nanoparticle” may refer to a nanoparticle that issubstantially free of a nucleotide or a polypeptide. As used herein, an“empty nanoparticle” or an “empty lipid nanoparticle” may refer to ananoparticle that consists substantially of only lipid components. Asused herein, a “loaded nanoparticle” or a “loaded lipid nanoparticle”(also referred to as a “full nanoparticle” or a “full lipidnanoparticle”) may refer to a nanoparticle comprising the components ofthe empty nanoparticle, and a therapeutic or prophylactic agent. As usedherein, a “loaded nanoparticle” or a “loaded lipid nanoparticle” (alsoreferred to as a “full nanoparticle” or a “full lipid nanoparticle”) mayrefer to a nanoparticle comprising the components of the emptynanoparticle, and a nucleotide or polypeptide. As used herein, a “loadednanoparticle” or a “loaded lipid nanoparticle” (also referred to as a“full nanoparticle” or a “full lipid nanoparticle”) may refer to ananoparticle comprising the components of the empty nanoparticle, and anucleic acid.

As used herein, “expression” of a nucleic acid sequence refers totranslation of an mRNA into a polypeptide or protein and/orpost-translational modification of a polypeptide or protein.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe or cell or tissue thereof).

As used herein, the term “ex vivo” refers to events that occur outsideof an organism (e.g., animal, plant, or microbe or cell or tissuethereof). Ex vivo events may take place in an environment minimallyaltered from a natural (e.g., in vivo) environment.

As used herein, the term “isomer” means any geometric isomer, tautomer,zwitterion, stereoisomer, enantiomer, or diastereomer of a compound.Compounds may include one or more chiral centers and/or double bonds andmay thus exist as stereoisomers, such as double-bond isomers (i.e.,geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or(−)) or cis/trans isomers). The present disclosure encompasses any andall isomers of the compounds described herein, including stereomericallypure forms (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereomeric mixtures of compounds andmeans of resolving them into their component enantiomers orstereoisomers are well-known.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertible by tautomerization is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (-CHO) in a sugar chain molecule reacting with one of thehydroxy groups (-OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of tautomerism in di-substituted guanidineis shown below.

It is to be understood that the compounds of the disclosure may bedepicted as different tautomers. It should also be understood that whencompounds have tautomeric forms, all tautomeric forms are intended to beincluded in the scope of the disclosure, and the naming of the compoundsdoes not exclude any tautomer form.

As used herein, a “lipid component” is that component of a nanoparticlecomposition that includes one or more lipids. For example, the lipidcomponent may include one or more cationic/ionizable, PEGylated,structural, or other lipids, such as phospholipids.

As used herein, a “linker” is a moiety connecting two moieties, forexample, the connection between two nucleosides of a cap species. Alinker may include one or more groups including but not limited tophosphate groups (e.g., phosphates, boranophosphates, thiophosphates,selenophosphates, and phosphonates), alkyl groups, amidates, orglycerols. For example, two nucleosides of a cap analog may be linked attheir 5′ positions by a triphosphate group or by a chain including twophosphate moieties and a boranophosphate moiety.

As used herein, “methods of administration” may include intravenous,intramuscular, intradermal, subcutaneous, or other methods of deliveringa composition to a subject. A method of administration may be selectedto target delivery (e.g., to specifically deliver) to a specific regionor system of a body.

As used herein, “modified” means non-natural. For example, an RNA may bea modified RNA. That is, an RNA may include one or more nucleobases,nucleosides, nucleotides, or linkers that are non-naturally occurring. A“modified” species may also be referred to herein as an “altered”species. Species may be modified or altered chemically, structurally, orfunctionally. For example, a modified nucleobase species may include oneor more substitutions that are not naturally occurring.

As used herein, the “N:P ratio” is the molar ratio of ionizable (in thephysiological pH range) nitrogen atoms in a lipid to phosphate groups inan RNA, e.g., in a nanoparticle composition including a lipid componentand an RNA.

As used herein, a “nanoparticle composition” is a composition comprisingone or more lipids. Nanoparticle compositions are typically sized on theorder of micrometers or smaller and may include a lipid bilayer.Nanoparticle compositions encompass lipid nanoparticles (LNPs),liposomes (e.g., lipid vesicles), and lipoplexes. For example, ananoparticle composition may be a liposome having a lipid bilayer with adiameter of 500 nm or less.

As used herein, “naturally occurring” means existing in nature withoutartificial aid.

As used herein, “patient” refers to a subject who may seek or be in needof treatment, requires treatment, is receiving treatment, will receivetreatment, or a subject who is under care by a trained professional fora particular disease or condition.

As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipidcomprising a polyethylene glycol component.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable excipient,” as used herein,refers to any ingredient other than the compounds described herein (forexample, a vehicle capable of suspending, complexing, or dissolving theactive compound) and having the properties of being substantiallynontoxic and non-inflammatory in a patient. Excipients may include, forexample: anti-adherents, antioxidants, binders, coatings, compressionaids, disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspending or dispersing agents, sweeteners, and waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A,vitamin E (alpha-tocopherol), vitamin C, xylitol, and other speciesdisclosed herein.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent disclosure includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like, it being understood that not all isomers mayhave the same level of activity. In addition, a crystal polymorphism maybe present for the compounds represented by the formula. It is notedthat any crystal form, crystal form mixture, or anhydride or hydratethereof is included in the scope of the present disclosure.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Compositions may also include salts of one or more compounds. Salts maybe pharmaceutically acceptable salts. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is altered by converting an existing acid orbase moiety to its salt form (e.g., by reacting a free base group with asuitable organic acid). Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. Representative acid addition saltsinclude acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred.

As used herein, a “phospholipid” is a lipid that includes a phosphatemoiety and one or more carbon chains, such as unsaturated fatty acidchains. A phospholipid may include one or more multiple (e.g., double ortriple) bonds (e.g., one or more unsaturations). Particularphospholipids may facilitate fusion to a membrane. For example, acationic phospholipid may interact with one or more negatively chargedphospholipids of a membrane (e.g., a cellular or intracellularmembrane). Fusion of a phospholipid to a membrane may allow one or moreelements of a lipid-containing composition to pass through the membranepermitting, e.g., delivery of the one or more elements to a cell.

As used herein, the “polydispersity index,” or “PDI” is a ratio thatdescribes the homogeneity of the particle size distribution of a system.A small value, e.g., less than 0.3, indicates a narrow particle sizedistribution.

As used herein, the term “polypeptide” or “polypeptide of interest”refers to a polymer of amino acid residues typically joined by peptidebonds that can be produced naturally (e.g., isolated or purified) orsynthetically. The terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer can comprise modified amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids such as homocysteine, ornithine, p-acetylphenylalanine,D-amino acids, and creatine), as well as other modifications known inthe art. The term, as used herein, refers to proteins, polypeptides, andpeptides of any size, structure, or function. Polypeptides includeencoded polynucleotide products, naturally occurring polypeptides,synthetic polypeptides, homologs, orthologs, paralogs, fragments andother equivalents, variants, and analogs of the foregoing. A polypeptidecan be a monomer or can be a multi-molecular complex such as a dimer,trimer or tetramer. They can also comprise single chain or multichainpolypeptides. Most commonly disulfide linkages are found in multichainpolypeptides. The term polypeptide can also apply to amino acid polymersin which one or more amino acid residues are an artificial chemicalanalogue of a corresponding naturally occurring amino acid. In someembodiments, a “peptide” can be less than or equal to 50 amino acidslong, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acidslong.

As used herein, an “RNA” refers to a ribonucleic acid that may benaturally or non-naturally occurring. For example, an RNA may includemodified and/or non-naturally occurring components such as one or morenucleobases, nucleosides, nucleotides, or linkers. An RNA may include acap structure, a chain terminating nucleoside, a stem loop, a polyAsequence, and/or a polyadenylation signal. An RNA may have a nucleotidesequence encoding a polypeptide of interest.

As used herein, a “DNA” refers to a desoxyribonucleic acid that may benaturally or non-naturally occurring. For example, a DNA may be asynthetic molecule, e.g., a synthetic DNA molecule produced in vitro. Insome embodiments, the DNA molecule is a recombinant molecule. As usedherein, a “recombinant DNA molecule” refers to a DNA molecule that doesnot exist as a natural product, but is produced using molecular biologytechniques.

As used herein, a “single unit dose” is a dose of any therapeuticadministered in one dose/at one time/single route/single point ofcontact, i.e., single administration event.

As used herein, a “split dose” is the division of single unit dose ortotal daily dose into two or more doses.

As used herein, a “total daily dose” is an amount given or prescribed in24 hour period. It may be administered as a single unit dose.

As used herein, “size” or “mean size” in the context of lipidnanoparticles (e.g., empty LNPs or loaded LNPs) refers to the meandiameter of a nanoparticle composition.

As used herein, the term “subject” or “patient” refers to any organismto which a composition in accordance with the disclosure may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

As used herein, “targeted cells” refers to any one or more cells ofinterest. The cells may be found in vitro, in vivo, in situ, or in thetissue or organ of an organism. The organism may be an animal,preferably a mammal, more preferably a human and most preferably apatient.

As used herein “target tissue” refers to any one or more tissue types ofinterest in which the delivery of a therapeutic and/or prophylacticwould result in a desired biological and/or pharmacological effect.Examples of target tissues of interest include specific tissues, organs,and systems or groups thereof. In particular applications, a targettissue may be a kidney, a lung, a spleen, vascular endothelium invessels (e.g., intra-coronary or intra-femoral), or tumor tissue (e.g.,via intratumoral injection). An “off-target tissue” refers to any one ormore tissue types in which the expression of the encoded protein doesnot result in a desired biological and/or pharmacological effect. Inparticular applications, off-target tissues may include the liver andthe spleen.

The term “therapeutic agent” or “prophylactic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect. Therapeutic agents are also referred to as“actives” or “active agents.” Such agents include, but are not limitedto, cytotoxins, radioactive ions, chemotherapeutic agents, smallmolecule drugs, proteins, and nucleic acids.

As used herein, the term “therapeutically effective amount” means anamount of an agent to be delivered (e.g., nucleic acid, drug,composition, therapeutic agent, diagnostic agent, prophylactic agent,etc.) that is sufficient, when administered to a subject suffering fromor susceptible to an infection, disease, disorder, and/or condition, totreat, improve symptoms of, diagnose, prevent, and/or delay the onset ofthe infection, disease, disorder, and/or condition.

As used herein, “transfection” refers to the introduction of a species(e.g., an RNA) into a cell. Transfection may occur, for example, invitro, ex vivo, or in vivo.

As used herein, the term “treating” refers to partially or completelyalleviating, ameliorating, improving, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of a particular infection,disease, disorder, and/or condition. For example, “treating” cancer mayrefer to inhibiting survival, growth, and/or spread of a tumor.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition and/or to a subject who exhibitsonly early signs of a disease, disorder, and/or condition for thepurpose of decreasing the risk of developing pathology associated withthe disease, disorder, and/or condition.

As used herein, the “zeta potential” is the electrokinetic potential ofa lipid, e.g., in a particle composition.

Nanoparticle Compositions

The disclosure also features lipid nanoparticles comprising a compoundaccording to (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb) asdescribed herein.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 um or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter), e.g., when measured by dynamic light scattering (DLS),transmission electron microscopy, scanning electron microscopy, oranother method. Nanoparticle compositions include, for example, lipidnanoparticles (LNPs; e.g., empty LNPs or loaded LNPs), liposomes, lipidvesicles, and lipoplexes. In some embodiments, nanoparticle compositionsare vesicles including one or more lipid bilayers. In certainembodiments, a nanoparticle composition includes two or more concentricbilayers separated by aqueous compartments. Lipid bilayers may befunctionalized and/or cross-linked to one another. Lipid bilayers mayinclude one or more ligands, proteins, or channels.

Nanoparticle compositions comprise a lipid component including at leastone compound according to Formula (A), (A1), (A2), (A3), (B), (I), (I′),(I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),(II), (IIa), or (IIb). For example, the lipid component of ananoparticle composition may include one or more of compounds ofTable 1. Nanoparticle compositions may also include a variety of othercomponents. For example, the lipid component of a nanoparticlecomposition may include one or more other lipids in addition to a lipidaccording to Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X),(I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II),(IIa), or (IIb).

Cationic/Ionizable Lipids

The lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may includeone or more cationic and/or ionizable lipids (e.g., lipids that may havea positive or partial positive charge at physiological pH) in additionto a lipid according to Formula (A), (A1), (A2), (A3), (B), (I), (I′),(I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),(II), (IIa), or (IIb).

Cationic and/or ionizable lipids may be selected from the non-limitinggroup consisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-{8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). In addition to these, a cationic lipid may also bea lipid including a cyclic amine group.

Structural Lipids

The lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may includeone or more structural lipids. Structural lipids can be selected fromthe group consisting of, but are not limited to, cholesterol,fecosterol, sitosterol, ergosterol, campesterol, stigmasterol,brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol,and mixtures thereof. In some embodiments, the structural lipid ischolesterol. In some embodiments, the structural lipid includescholesterol and a corticosteroid (such as prednisolone, dexamethasone,prednisone, and hydrocortisone), or a combination thereof. In someembodiments, the structural lipid is:

Phospholipids

The lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may includeone or more phospholipids, such as one or more (poly)unsaturated lipids.Phospholipids may assemble into one or more lipid bilayers. In general,phospholipids may include a phospholipid moiety and one or more fattyacid moieties. For example, a phospholipid may be a lipid according toFormula (IV):

in which R_(p) represents a phospholipid moiety and RA and RB representfatty acid moieties with or without unsaturation that may be the same ordifferent. A phospholipid moiety may be selected from the non-limitinggroup consisting of phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid,2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety maybe selected from the non-limiting group consisting of lauric acid,myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucicacid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoicacid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.Non-natural species including natural species with modifications andsubstitutions including branching, oxidation, cyclization, and alkynesare also contemplated. For example, a phospholipid may be functionalizedwith or cross-linked to one or more alkynes (e.g., an alkenyl group inwhich one or more double bonds is replaced with a triple bond). Underappropriate reaction conditions, an alkyne group may undergo acopper-catalyzed cycloaddition upon exposure to an azide. Such reactionsmay be useful in functionalizing a lipid bilayer of a lipid nanoparticle(e.g., an empty LNP or a loaded LNP) to facilitate membrane permeationor cellular recognition or in conjugating a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) to a useful component such as a targeting orimaging moiety (e.g., a dye).

Phospholipids useful in the compositions and methods may be selectedfrom the non-limiting group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),dipalmitoylphosphatidylglycerol (DPPG),palmitoyloleoylphosphatidylethanolamine (POPE),distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine (LPE), and mixtures thereof. In someembodiments, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP)includes DSPC. In certain embodiments, a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) includes DOPE. In some embodiments, a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) includes both DSPC andDOPE.

PEG Lipids

The lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may includeone or more PEG or PEG-modified lipids. Such species may be alternatelyreferred to as PEGylated lipids. A PEG lipid is a lipid modified withpolyethylene glycol. A PEG lipid may be selected from the non-limitinggroup consisting of PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modifieddialkylamines, PEG-modified diacylglycerols (PEG-DEG), PEG-modifieddialkylglycerols, and mixtures thereof. For example, a PEG lipid may bePEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In certain embodiments, the PEG lipid is selected from the groupconsisting of a PEG-modified phosphatidylethanolamine, a PEG-modifiedphosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine,a PEG-modified diacylglycerol, and a PEG-modified dialkylglycerol.

In certain embodiments, PEG lipid is selected from the group consistingof 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). For example, in someembodiments, the PEG lipid is PEG-DMG.

In certain embodiments, the PEG lipid is a compound of Formula (PL-I):

or a salt thereof, wherein:

R^(3PL1) is —OR^(OPL1);

R^(OPL1) is hydrogen, optionally substituted alkyl, or an oxygenprotecting group;

r^(PL1) is an integer between 1 and 100, inclusive;

L¹ is optionally substituted C₁₋₁₀ alkylene, wherein at least onemethylene of the optionally substituted C₁₋₁₀ alkylene is independentlyreplaced with optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted arylene, optionallysubstituted heteroarylene, O, N(R^(NPL1)), S, C(O), C(O)N(R^(NPL1)),NR^(NPL1)C(O), —C(O)O, OC(O), OC(O)O, OC(O)N(R^(NPL1)), NR^(NPL1)C(O)O,or NR^(NPL1)C(O)N(R^(NPL1));

D is a moiety obtained by click chemistry or a moiety cleavable underphysiological conditions;

m^(PL1) is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

each instance of of L² is independently a bond or optionally substitutedC₁₋₆ alkylene, wherein one methylene unit of the optionally substitutedC₁₋₆ alkylene is optionally replaced with O, N(R^(NPL1)), S, C(O),C(O)N(R^(NPL1)), NR^(NPL1)C(O), C(O)O, OC(O), OC(O)O, —OC(O)N(R^(NPL1)),NR^(NPL1)C(O)O, or NR^(NPL1)C(O)N(R^(NPL1));

each instance of R^(2SL) is independently optionally substituted C₁₋₃₀alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally substitutedC₁₋₃₀ alkynyl; optionally wherein one or more methylene units of R^(2SL)are independently replaced with optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,optionally substituted heteroarylene, N(R^(NPL1)), O, S, C(O),C(O)N(R^(NPL1)), NR^(NPL1)C(O), —NR^(NPL1)C(O)N(R^(NPL1)), C(O)O, OC(O),OC(O)O, OC(O)N(R^(NPL1)), NR^(NPL1)C(O)O, C(O)S, —SC(O), C(═NR^(NPL1)),C(═NR^(NPL1))N(R^(NPL1)), NR^(NPL1)C(═NR^(NPL1)),—NR^(NPL1)C(═NR^(NPL1))N(R^(NPL1)), C(S), C(S)N(R^(NPL1)),NR^(NPL1)C(S), NR^(NPL1)C(S)N(R^(NPL1)), S(O) OS(O), S(O)O, OS(O)O,OS(O)₂, S(O)₂O, OS(O)₂O, N(R^(NPL1))S(O), S(O)N(R^(NPL1)),—N(R^(NPL1))S(O)N(R^(NPL1)), OS(O)N(R^(NPL1)), N(R^(NPL1))S(O)O, S(O)₂,N(R^(NPL1))S(O)₂, —S(O)₂N(R^(NPL1)), N(R^(NPL1))S(O)₂N(R^(NPL1)),OS(O)₂N(R^(NPL1)), or N(R^(NPL1))S(O)₂O;

each instance of R^(NPL1) is independently hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl; and

p^(SL) is 1 or 2.

In certain embodiments, the PEG lipid is a compound of Formula(PL-I-OH):

or a salt thereof.

In certain embodiments, the PEG lipid is a compound of Formula(PL-II-OH):

or a salt or isomer thereof, wherein:

R^(3PEG) is-OR^(O);

R^(O) is hydrogen, C₁₋₆ alkyl or an oxygen protecting group;

r^(PEG) is an integer between 1 and 100;

R^(5PEG) is C₁₀₋₄₀ alkyl, C₁₀₋₄₀ alkenyl, or C₁₀₋₄₀ alkynyl; andoptionally one or more methylene groups of R^(5PEG) are independentlyreplaced with C₃₋₁₀ carbocyclylene, 4 to 10 membered heterocyclylene,C₆₋₁₀ arylene, 4 to 10 membered heteroarylene, —N(R^(NPEG))—, —O—, —S—,—C(O)—, —C(O)N(R^(NPEG))—, —NR^(NPEG)C(O)—, —NR^(NPEG)C(O)N(R^(NPEG))—,—C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(NPEG))—, —NR^(NPEG)C(O)O—,—C(O)S—, —SC(O)—, —C(═NR^(NPEG))—, —C(═NR^(NPEG))N(R^(NPEG))—,—NR^(NPEG)C(═NR^(NPEG))—,—NR^(NPEG)C(═NR^(NPEG))N(R^(NPEG))—, —C(S)—,—C(S)N(R^(NPEG))—, —NR^(NPEG)C(S)—, —NR^(NPEG)C(S)N(R^(NPEG))—, —OS(O)—,—S(O)O—, —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—, —N(R^(NPEG))S(O)—,—S(O)N(R^(NPEG))—, —N(R^(NPEG))S(O)N(R^(NPEG))—, —OS(O)N(R^(NPEG))—,—N(R^(NPEG))S(O)O—, —S(O)₂—, —N(R^(NPEG))S(O)₂—, —S(O)₂N(R^(NPEG))—,—N(R^(NPEG))S(O)₂N(R^(NPEG))—, —OS(O)₂N(R^(NPEG))—, or—N(R^(NPEG))S(O)₂O—; and

each instance of R^(NPEG) is independently hydrogen, C₁₋₆ alkyl, or anitrogen protecting group.

In certain embodiments, in the PEG lipid of Formula (PL-II-OH), r is aninteger between 40 and 50. For example, r is selected from the groupconsinsting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. Forexample, r is 45.

In certain embodiments, in the PEG lipid of Formula (PL-II-OH), R⁵ isC₁₇ alkyl.

In certain embodiments, the PEG lipid is a compound of Formula (PL-II):

wherein r^(PEG) is an integer between 1 and 100.

In certain embodiments, the PEG lipid is a compound of Formula (PEG-1):

In certain embodiments, the PEG lipid is a compound of Formula (PL-III):

or a salt or isomer thereof, wherein S^(PL1) is an integer between 1 and100.

In certain embodiments, the PEG lipid is a compound of followingformula:

In certain embodiments, the incorporation of lipids of one of formulae(PL-I), PL-I-OH), (PL-II), (PL-II-OH), (PL-III), PEG_(2k)-DMG, or PEG-1in the nanoparticle formulation can improve the pharmacokinetics and/orbiodistribution of the lipid nanoparticle formulations. For example,incorporation of lipids of one of formulae (PL-II-OH), (PL-IIa-OH),(PL-II), or PEG-lin the nanoparticle formulation can reduce theaccelerated blood clearance (ABC) effect.

Adjuvants

In some embodiments, a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) that includes one or more lipids described herein mayfurther include one or more adjuvants, e.g., Glucopyranosyl LipidAdjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B),poly(I:C), aluminum hydroxide, and Pam3CSK4.

Therapeutic Agents

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may include one ormore therapeutic and/or prophylactics. The disclosure features methodsof delivering a therapeutic and/or prophylactic to a mammalian cell ororgan, producing a polypeptide of interest in a mammalian cell, andtreating a disease or disorder in a mammal in need thereof comprisingadministering to a mammal and/or contacting a mammalian cell with alipid nanoparticle (e.g., an empty LNP or a loaded LNP) including atherapeutic and/or prophylactic.

Therapeutic and/or prophylactics include biologically active substancesand are alternately referred to as “active agents.” A therapeutic and/orprophylactic may be a substance that, once delivered to a cell or organ,brings about a desirable change in the cell, organ, or other bodilytissue or system. Such species may be useful in the treatment of one ormore diseases, disorders, or conditions. In some embodiments, atherapeutic and/or prophylactic is a small molecule drug useful in thetreatment of a particular disease, disorder, or condition.

In some embodiments, a therapeutic and/or prophylactic is a vaccine, acompound (e.g., a polynucleotide or nucleic acid molecule that encodes aprotein or polypeptide or peptide or a protein or polypeptide orprotein) that elicits an immune response, and/or another therapeuticand/or prophylactic. Vaccines include compounds and preparations thatare capable of providing immunity against one or more conditions relatedto infectious diseases and can include mRNAs encoding infectious diseasederived antigens and/or epitopes.

Vaccines also include compounds and preparations that direct an immuneresponse against cancer cells and can include mRNAs encoding tumor cellderived antigens, epitopes, and/or neoepitopes. In some embodiments, avaccine and/or a compound capable of eliciting an immune response isadministered intramuscularly via a composition of the disclosure.

In other embodiments, a therapeutic and/or prophylactic is a protein,for example a protein needed to augment or replace a naturally-occurringprotein of interest. Such proteins or polypeptides may be naturallyoccurring, or may be modified using methods known in the art, e.g., toincrease half life. Exemplary proteins are intracellular, transmembrane,or secreted.

Polynucleotides and Nucleic Acids

In some embodiments, the therapeutic agent is an agent that enhances(i.e., increases, stimulates, upregulates) protein expression.Non-limiting examples of types of therapeutic agents that can be usedfor enhancing protein expression include RNAs, mRNAs, dsRNAs,CRISPR/Cas9 technology, ssDNAs and DNAs (e.g., expression vectors). Theagent that upregulates protein expression may upregulate expression of anaturally occurring or non-naturally occurring protein (e.g., a chimericprotein that has been modified to improve half life, or one thatcomprises desirable amino acid changes). Exemplary proteins includeintracellular, transmembrane, or secreted proteins, peptides, orpolypeptides.

In some embodiments, the therapeutic agent is a DNA therapeutic agent.The DNA molecule can be a double-stranded DNA, a single-stranded DNA(ssDNA), or a molecule that is a partially double-stranded DNA, i.e.,has a portion that is double-stranded and a portion that issingle-stranded. In some cases the DNA molecule is triple-stranded or ispartially triple-stranded, i.e., has a portion that is triple strandedand a portion that is double stranded. The DNA molecule can be acircular DNA molecule or a linear DNA molecule.

A DNA therapeutic agent can be a DNA molecule that is capable oftransferring a gene into a cell, e.g., that encodes and can express atranscript. In other embodiments, the

DNA molecule is a synthetic molecule, e.g., a synthetic DNA moleculeproduced in vitro. In some embodiments, the DNA molecule is arecombinant molecule. Non-limiting exemplary DNA therapeutic agentsinclude plasmid expression vectors and viral expression vectors.

The DNA therapeutic agents described herein, e.g., DNA vectors, caninclude a variety of different features. The DNA therapeutic agentsdescribed herein, e.g., DNA vectors, can include a non-coding DNAsequence. For example, a DNA sequence can include at least oneregulatory element for a gene, e.g., a promoter, enhancer, terminationelement, polyadenylation signal element, splicing signal element, andthe like. In some embodiments, the non-coding DNA sequence is an intron.In some embodiments, the non-coding DNA sequence is a transposon. Insome embodiments, a DNA sequence described herein can have a non-codingDNA sequence that is operatively linked to a gene that istranscriptionally active. In other embodiments, a DNA sequence describedherein can have a non-coding DNA sequence that is not linked to a gene,i.e., the non-coding DNA does not regulate a gene on the DNA sequence.

In some embodiments, in the loaded LNP of the disclosure, the one ormore therapeutic and/or prophylactic agents is a nucleic acid. In someembodiments, the one or more therapeutic and/or prophylactic agents isselected from the group consisting of a ribonucleic acid (RNA) and adeoxyribonucleic acid (DNA).

For example, in some embodiments, when the therapeutic and/orprophylactic agents is a DNA, the DNA is selected from the groupconsisting of a double-stranded DNA, a single-stranded DNA (ssDNA), apartially double-stranded DNA, a triple stranded DNA, and a partiallytriple-stranded DNA. In some embodiments, the DNA is selected from thegroup consisting of a circular DNA, a linear DNA, and mixtures thereof.

In some embodiments, in the loaded LNP of the disclosure, the one ormore therapeutic and/or prophylactic agents is selected from the groupconsisting of a plasmid expression vector, a viral expression vector,and mixtures thereof.

For example, in some embodiments, when the therapeutic and/orprophylactic agents is a RNA, the RNA is selected from the groupconsisting of a single-stranded RNA, a double-stranded RNA (dsRNA), apartially double-stranded RNA, and mixtures thereof. In someembodiments, the RNA is selected from the group consisting of a circularRNA, a linear RNA, and mixtures thereof.

For example, in some embodiments, when the therapeutic and/orprophylactic agents is a RNA, the RNA is selected from the groupconsisting of a short interfering RNA (siRNA), an asymmetricalinterfering RNA (aiRNA), a RNA interference (RNAi) molecule, a microRNA(miRNA), an antagomir, an antisense RNA, a ribozyme, a Dicer-substrateRNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), lockednucleic acids (LNAs) and CRISPR/Cas9 technology, and mixtures thereof.

For example, in some embodiments, when the therapeutic and/orprophylactic agents is a RNA, the RNA is selected from the groupconsisting of a small interfering RNA (siRNA), an asymmetricalinterfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA(dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), andmixtures thereof.

In some embodiments, the one or more therapeutic and/or prophylacticagents is an mRNA. In some embodiments, the one or more therapeuticand/or prophylactic agents is a modified mRNA (mmRNA).

In some embodiments, the one or more therapeutic and/or prophylacticagents is an mRNA that incorporates a micro-RNA binding site (miRbinding site). Further, in some embodiments, an mRNA includes one ormore of a stem loop, a chain terminating nucleoside, a polyA sequence, apolyadenylation signal, and/or a 5′ cap structure.

An mRNA may be a naturally or non-naturally occurring mRNA. An mRNA mayinclude one or more modified nucleobases, nucleosides, or nucleotides,as described below, in which case it may be referred to as a “modifiedmRNA” or “mmRNA.” As described herein “nucleoside” is defined as acompound containing a sugar molecule (e.g., a pentose or ribose) orderivative thereof in combination with an organic base (e.g., a purineor pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group.

An mRNA may include a 5′ untranslated region (5′-UTR), a 3′ untranslatedregion (3′-UTR), and/or a coding region (e.g., an open reading frame).An mRNA may include any suitable number of base pairs, including tens(e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100), hundreds (e.g., 200,300, 400, 500, 600, 700, 800, or 900) or thousands (e.g., 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Anynumber (e.g., all, some, or none) of nucleobases, nucleosides, ornucleotides may be an analog of a canonical species, substituted,modified, or otherwise non-naturally occurring. In certain embodiments,all of a particular nucleobase type may be modified. In someembodiments, all uracils or uridines are modified. When all nucleobases,nucleosides, or nucleotides are modified, e.g., all uracils or uridines,the mRNA can be referred to as “fully modified”, e.g., for uracil oruridine.

In some embodiments, an mRNA as described herein may include a 5′ capstructure, a chain terminating nucleotide, optionally a Kozak sequence(also known as a Kozak consensus sequence), a stem loop, a polyAsequence, and/or a polyadenylation signal.

A 5′ cap structure or cap species is a compound including two nucleosidemoieties joined by a linker and may be selected from a naturallyoccurring cap, a non-naturally occurring cap or cap analog, or ananti-reverse cap analog (ARCA). A cap species may include one or moremodified nucleosides and/or linker moieties. For example, a natural mRNAcap may include a guanine nucleotide and a guanine (G) nucleotidemethylated at the 7 position joined by a triphosphate linkage at their5′ positions, e.g., m7G(5′)ppp(5′)G, commonly written as m7GpppG. A capspecies may also be an anti-reverse cap analog. A non-limiting list ofpossible cap species includes m7GpppG, m7Gpppm7G, m73′dGpppG,m27,O3′GpppG, m27,O3′GppppG, m27,O2′GppppG, m7Gpppm7G, m73′dGpppG,m27,O3′GpppG, m27,O3′GppppG, and m27,O2′GppppG.

An mRNA may instead or additionally include a chain terminatingnucleoside. For example, a chain terminating nucleoside may includethose nucleosides deoxygenated at the 2′ and/or 3′ positions of theirsugar group. Such species may include 3′ deoxyadenosine (cordycepin), 3′deoxyuridine, 3′ deoxycytosine, 3′ deoxyguanosine, 3′ deoxythymine, and2′,3′ dideoxynucleosides, such as 2′,3′ dideoxyadenosine, 2′,3′dideoxyuridine, 2′,3′ dideoxycytosine, 2′,3′ dideoxyguanosine, and 2′,3′dideoxythymine. In some embodiments, incorporation of a chainterminating nucleotide into an mRNA, for example at the 3′-terminus, mayresult in stabilization of the mRNA.

An mRNA may instead or additionally include a stem loop, such as ahistone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or morenucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7,or 8 nucleotide base pairs. A stem loop may be located in any region ofan mRNA. For example, a stem loop may be located in, before, or after anuntranslated region (a 5′ untranslated region or a 3′ untranslatedregion), a coding region, or a polyA sequence or tail. In someembodiments, a stem loop may affect one or more function(s) of an mRNA,such as initiation of translation, translation efficiency, and/ortranscriptional termination.

An mRNA may instead or additionally include a polyA sequence and/orpolyadenylation signal. A polyA sequence may be comprised entirely ormostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may also comprise stabilizing nucleotides or analogs. Forexample, a poly A sequence can include deoxythymidine, e.g., inverted(or reverse linkage) deoxythymidine (dT), as a stabilizing nucleotide oranalog. Detials on using inverted dT and other stabilizing poly Asequence modifications can be found, for example, in WO2017/049275 A2,the content of which is incoported herein by reference. A polyA sequencemay be a tail located adjacent to a 3′ untranslated region of an mRNA.In some embodiments, a polyA sequence may affect the nuclear export,translation, and/or stability of an mRNA.

An mRNA may instead or additionally include a microRNA binding site.MicroRNA binding sites (or miR binding sites) can be used to regulatemRNA expression in various tissues or cell types. In exemplaryembodiments, miR binding sites are engineered into 3′ UTR sequences ofan mRNA to regulate, e.g., enhance degradation of mRNA in cells ortissues expressing the cognate miR. Such regulation is useful toregulate or control “off-target” expression it mRNAs, i.e., expressionin undesired cells or tissues in vivo. Detials on using mir bindingsites can be found, for example, in WO 2017/062513 A2, the content ofwhich is incoported herein by reference.

In some embodiments, an mRNA is a bicistronic mRNA comprising a firstcoding region and a second coding region with an intervening sequencecomprising an internal ribosome entry site (IRES) sequence that allowsfor internal translation initiation between the first and second codingregions, or with an intervening sequence encoding a self-cleavingpeptide, such as a 2A peptide. IRES sequences and 2A peptides aretypically used to enhance expression of multiple proteins from the samevector. A variety of IRES sequences are known and available in the artand may be used, including, e.g., the encephalomyocarditis virus IRES.

In some embodiments, an mRNA of the disclosure comprises one or moremodified nucleobases, nucleosides, or nucleotides (termed “modifiedmRNAs” or “mmRNAs”). In some embodiments, modified mRNAs may have usefulproperties, including enhanced stability, intracellular retention,enhanced translation, and/or the lack of a substantial induction of theinnate immune response of a cell into which the mRNA is introduced, ascompared to a reference unmodified mRNA. Therefore, use of modifiedmRNAs may enhance the efficiency of protein production, intracellularretention of nucleic acids, as well as possess reduced immunogenicity.

In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3 or 4)different modified nucleobases, nucleosides, or nucleotides. In someembodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modifiednucleobases, nucleosides, or nucleotides. In some embodiments, themodified mRNA may have reduced degradation in a cell into which the mRNAis introduced, relative to a corresponding unmodified mRNA.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U),4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U),5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U),5-carboxyhydroxymethyl-uridine methyl ester (mchm5U),5-methoxycarbonylmethyl-uridine (mcm5U),5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U),5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine(mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U),5-methylaminomethyl-2-seleno-uridine (mnm5se2U),5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine(cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm5U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm5s2U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ),5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp3 ψ),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 yr),5-(isopentenylaminomethyl)uridine (inmSU),5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′—O-dimethyl-uridine (m5Um),2′-O-methyl-pseudouridine (kvm), 2-thio-2′-O-methyl-uridine (s2Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um),5-carboxymethylaminomethyl-2′—O—methyl-uridine (cmnm5Um),3,2′—O-dimethyl-uridine (m3Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C),N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′4)-dimethyl-cytidine (m5Cm), N4-acetyl-2′-O-methyl-cytidine (ac4Cm),N4,2′—O-dimethyl-cytidine (m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm),N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includea-thio-adenosine, 2-amino-purine, 2, 6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A),2-methyl-adenine (m2A), N6-methyl-adenosine (m6A),2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine(i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A),N6-(cis-hydroxyisopentenyl)adenosine (io6A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A),N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine(t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A),N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A),N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′—O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl-adenosine(m62Am), 1,2′—O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeα-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW),undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G),N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m2Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm),1-methyl-2′-O-methyl-guanosine (m1Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m1Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)) , 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In some embodiments, an mRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine ,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.) In some embodiments, themodified nucleobase is N1-methylpseudouridine (m1ψ) and the mRNA of thedisclosure is fully modified with N1-methylpseudouridine (m1ψ). In someembodiments, N1-methylpseudouridine (m1ψ) represents from 75-100% of theuracils in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ)represents 100% of the uracils in the mRNA.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine. In some embodiments, an mRNA of the disclosureincludes a combination of one or more of the aforementioned modifiednucleobases (e.g., a combination of 2, 3 or 4 of the aforementionedmodified nucleobases.)

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A),N6-methyl-adenosine (m6A). In some embodiments, an mRNA of thedisclosure includes a combination of one or more of the aforementionedmodified nucleobases (e.g., a combination of 2, 3 or 4 of theaforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. Insome embodiments, an mRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the mRNA comprises pseudouridine (ψ). In someembodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine(m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNAcomprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In someembodiments, the mRNA comprises 2′-O-methyl uridine. In someembodiments, the mRNA comprises 2′-O-methyl uridine and5-methyl-cytidine (m5C). In some embodiments, the mRNA comprisescomprises N6-methyl-adenosine (m6A). In some embodiments, the mRNAcomprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In certain embodiments, an mRNA of the disclosure is uniformly modified(i.e., fully modified, modified through-out the entire sequence) for aparticular modification. For example, an mRNA can be uniformly modifiedwith N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), meaningthat all uridines or all cytosine nucleosides in the mRNA sequence arereplaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C).Similarly, mRNAs of the disclosure can be uniformly modified for anytype of nucleoside residue present in the sequence by replacement with amodified residue such as those set forth above.

In some embodiments, an mRNA of the disclosure may be modified in acoding region (e.g., an open reading frame encoding a polypeptide). Inother embodiments, an mRNA may be modified in regions besides a codingregion. For example, in some embodiments, a 5′-UTR and/or a 3′-UTR areprovided, wherein either or both may independently contain one or moredifferent nucleoside modifications. In such embodiments, nucleosidemodifications may also be present in the coding region.

The mmRNAs of the disclosure can include a combination of modificationsto the sugar, the nucleobase, and/or the intemucleoside linkage. Thesecombinations can include any one or more modifications described herein.

Where a single modification is listed, the listed nucleoside ornucleotide represents 100 percent of that A, U, G or C nucleotide ornucleoside having been modified. Where percentages are listed, theserepresent the percentage of that particular A, U, G or C nucleobasetriphosphate of the total amount of A, U, G, or C triphosphate present.For example, the combination: 25% 5-Aminoallyl-CTP+75% CTP/25%5-Methoxy-UTP+75% UTP refers to a polynucleotide where 25% of thecytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosinesare CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of theuracils are UTP. Where no modified UTP is listed then the naturallyoccurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of thosenucleotides found in the polynucleotide. In this example all of the GTPand ATP nucleotides are left unmodified.

The mRNAs of the present disclosure, or regions thereof, may be codonoptimized. Codon optimization methods are known in the art and may beuseful for a variety of purposes: matching codon frequencies in hostorganisms to ensure proper folding, bias GC content to increase mRNAstability or reduce secondary structures, minimize tandem repeat codonsor base runs that may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeproteins trafficking sequences, remove/add post translation modificationsites in encoded proteins (e.g., glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, adjust translationrates to allow the various domains of the protein to fold properly, orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art; non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park, Calif.) and/or proprietarymethods. In some embodiments, the mRNA sequence is optimized usingoptimization algorithms, e.g., to optimize expression in mammalian cellsor enhance mRNA stability.

In certain embodiments, the present disclosure includes polynucleotideshaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% sequence identity to any of the polynucleotidesequences described herein.

mRNAs of the present disclosure may be produced by means available inthe art, including but not limited to in vitro transcription (IVT) andsynthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combinedsynthetic methods, small region synthesis, and ligation methods may beutilized. In some embodiments, mRNAs are made using IVT enzymaticsynthesis methods. Accordingly, the present disclosure also includespolynucleotides, e.g., DNA, constructs and vectors that may be used toin vitro transcribe an mRNA described herein.

Non-natural modified nucleobases may be introduced into polynucleotides,e.g., mRNA, during synthesis or post-synthesis. In certain embodiments,modifications may be on internucleoside linkages, purine or pyrimidinebases, or sugar. In particular embodiments, the modification may beintroduced at the terminal of a polynucleotide chain or anywhere else inthe polynucleotide chain; with chemical synthesis or with a polymeraseenzyme.

Either enzymatic or chemical ligation methods may be used to conjugatepolynucleotides or their regions with different functional moieties,such as targeting or delivery agents, fluorescent labels, liquids,nanoparticles, etc. Therapeutic Agents for Reducing Protein Expression

In some embodiments, the therapeutic agent is a therapeutic agent thatreduces (i.e., decreases, inhibits, downregulates) protein expression.Non-limiting examples of types of therapeutic agents that can be usedfor reducing protein expression include mRNAs that incorporate amicro-RNA binding site(s) (miR binding site), microRNAs (miRNAs),antagomirs, small (short) interfering RNAs (siRNAs) (including shortmersand dicer-substrate RNAs), RNA interference (RNAi) molecules, antisenseRNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids(LNAs) and CRISPR/Cas9 technology.

Peptide/Polypeptide Therapeutic Agents

In some embodiments, the therapeutic agent is a peptide therapeuticagent. In some embodiments the therapeutic agent is a polypeptidetherapeutic agent.

In some embodiments, the peptide or polypeptide is naturally-derived,e.g., isolated from a natural source. In other embodiments, the peptideor polypeptide is a synthetic molecule, e.g., a synthetic peptide orpolypeptide produced in vitro. In some embodiments, the peptide orpolypeptide is a recombinant molecule. In some embodiments, the peptideor polypeptide is a chimeric molecule. In some embodiments, the peptideor polypeptide is a fusion molecule. In some embodiments, the peptide orpolypeptide therapeutic agent of the composition is a naturallyoccurring peptide or polypeptide. In some embodiments, the peptide orpolypeptide therapeutic agent of the composition is a modified versionof a naturally occurring peptide or polypeptide (e.g., contains lessthan 3, less than 5, less than 10, less than 15, less than 20, or lessthan 25 amino substitutions, deletions, or additions compared to itswild type, naturally occurring peptide or polypeptide counterpart).

In some embodiments, in the loaded LNP of the disclosure, the one ormore therapeutic and/or prophylactic agents is a polynucleotide or apolypeptide.

Other Components

A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may includeone or more components in addition to those described in the precedingsections. For example, a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may include one or more small hydrophobic molecules such asa vitamin (e.g., vitamin A or vitamin E) or a sterol.

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may also includeone or more permeability enhancer molecules, carbohydrates, polymers,surface altering agents, or other components. Carbohydrates may includesimple sugars (e.g., glucose) and polysaccharides (e.g., glycogen andderivatives and analogs thereof).

A polymer may be included in and/or used to encapsulate or partiallyencapsulate a nanoparticle composition. A polymer may be biodegradableand/or biocompatible. A polymer may be selected from, but is not limitedto, polyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,and polyarylates. For example, a polymer may include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP),polysiloxanes, polystyrene (PS), polyurethanes, derivatized cellulosessuch as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, hydroxypropylcellulose,carboxymethylcellulose, polymers of acrylic acids, such aspoly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate) and copolymers and mixtures thereof,polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylenefumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters,poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM),poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), andpolyglycerol.

Surface altering agents may include, but are not limited to, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g.,acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine,carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol,letosteine, stepronin, tiopronin, gelsolin, thymosin (34, dornase alfa,neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surfacealtering agent may be disposed within a nanoparticle and/or on thesurface of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP)(e.g., by coating, adsorption, covalent linkage, or other process).

A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may alsocomprise one or more functionalized lipids. For example, a lipid may befunctionalized with an alkyne group that, when exposed to an azide underappropriate reaction conditions, may undergo a cycloaddition reaction.In particular, a lipid bilayer may be functionalized in this fashionwith one or more groups useful in facilitating membrane permeation,cellular recognition, or imaging. The surface of a lipid nanoparticle(e.g., an empty LNP or a loaded LNP) may also be conjugated with one ormore useful antibodies. Functional groups and conjugates useful intargeted cell delivery, imaging, and membrane permeation are well knownin the art.

In addition to these components, lipid nanoparticles (e.g., empty LNPsor loaded LNPs) may include any substance useful in pharmaceuticalcompositions. For example, the lipid nanoparticle (e.g., an empty LNP ora loaded LNP) may include one or more pharmaceutically acceptableexcipients or accessory ingredients such as, but not limited to, one ormore solvents, dispersion media, diluents, dispersion aids, suspensionaids, granulating aids, disintegrants, fillers, glidants, liquidvehicles, binders, surface active agents, isotonic agents, thickening oremulsifying agents, buffering agents, lubricating agents, oils,preservatives, and other species. Excipients such as waxes, butters,coloring agents, coating agents, flavorings, and perfuming agents mayalso be included.

Examples of diluents may include, but are not limited to, calciumcarbonate, sodium carbonate, calcium phosphate, dicalcium phosphate,calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose,sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol,sorbitol, inositol, sodium chloride, dry starch, cornstarch, powderedsugar, and/or combinations thereof. Granulating and dispersing agentsmay be selected from the non-limiting list consisting of potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate,quaternary ammonium compounds, and/or combinations thereof.

Surface active agents and/or emulsifiers may include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or combinations thereof.

A binding agent may be starch (e.g. cornstarch and starch paste);gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses,lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia,sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilageof isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®),and larch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; and combinations thereof, or any other suitable bindingagent.

Examples of preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Examples of antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Examples ofchelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Examples of antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Examples of antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Examples of alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, benzyl alcohol, phenol, phenoliccompounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethylalcohol. Examples of acidic preservatives include, but are not limitedto, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, aceticacid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phyticacid. Other preservatives include, but are not limited to, tocopherol,tocopherol acetate, deteroxime mesylate, cetrimide, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine,sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodiumbisulfite, sodium metabisulfite, potassium sulfite, potassiummetabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115,GERMABEN®II, NEOLONETM, KATHONTM, and/or EUXYL®.

Examples of buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, calcium lactobionate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, and/or combinationsthereof. Lubricating agents may selected from the non-limiting groupconsisting of magnesium stearate, calcium stearate, stearic acid,silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, magnesium lauryl sulfate, sodium lauryl sulfate, andcombinations thereof.

Examples of oils include, but are not limited to, almond, apricotkernel, avocado, babassu, bergamot, black current seed, borage, cade,camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter,coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, eveningprimrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut,hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle,tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate,caprylic triglyceride, capric triglyceride, cyclomethicone, diethylsebacate, dimethicone 360, simethicone, isopropyl myristate, mineraloil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinationsthereof.

Formulations

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may include alipid component and one or more additional components, such as atherapeutic and/or prophylactic. A lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) may be designed for one or more specificapplications or targets. The elements of a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) may be selected based on a particularapplication or target, and/or based on the efficacy, toxicity, expense,ease of use, availability, or other feature of one or more elements.Similarly, the particular formulation of a nanoparticle composition maybe selected for a particular application or target according to, forexample, the efficacy and toxicity of particular combinations ofelements.

The lipid component of a nanoparticle composition may include, forexample, a lipid according to Formula (A), (A1), (A2), (A3), (B), (I),(I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If),(Ig), (II), (IIa), or (IIb), a phospholipid (such as an unsaturatedlipid, e.g., DOPE or DSPC), a PEG lipid, and a structural lipid. Theelements of the lipid component may be provided in specific fractions.

In some embodiments, the lipid component of a nanoparticle compositionincludes a lipid according to Formula (A), (A1), (A2), (A3), (B), (I),(I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If),(Ig), (II), (IIa), or (IIb), a phospholipid, a PEG lipid, and astructural lipid. In certain embodiments, the lipid component of thenanoparticle composition includes about 30 mol % to about 60 mol %compound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % toabout 48.5 mol % structural lipid, and about 0 mol % to about 10 mol %of PEG lipid, provided that the total mol % does not exceed 100%. Insome embodiments, the lipid component of the nanoparticle compositionincludes about 35 mol % to about 55 mol % compound of Formula (A), (A1),(A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic),(Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), about 5 mol % to about 25mol % phospholipid, about 30 mol % to about 40 mol % structural lipid,and about 0 mol % to about 10 mol % of PEG lipid. In a particularembodiment, the lipid component includes about 50 mol % said compound,about 10 mol % phospholipid, about 38.5 mol % structural lipid, andabout 1.5 mol % of PEG lipid. In another particular embodiment, thelipid component includes about 40 mol % said compound, about 20 mol %phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % ofPEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. Inother embodiments, the PEG lipid may be PEG-1, or PEG_(2k)-DMG and/orthe structural lipid may be cholesterol.

In some embodiments an empty lipid nanoparticle (empty LNP) comprises acompound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), a phospholipid, a structural lipid, and a PEG lipid.

In some embodiments a loaded lipid nanoparticle (loaded LNP) comprises acompound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), a phospholipid, a structural lipid, a PEG lipid, and one or moretherapeutic and/or prophylactic agents.

In some embodiments, the empty LNP or loaded LNP comprises the compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),in an amount from about 40% to about 60%.

In some embodiments, the empty LNP or loaded LNP comprises thephospholipid in an amount from about 0% to about 20%. For example, insome embodiments, the empty LNP or loaded LNP comprises DSPC in anamount from about 0% to about 20%.

In some embodiments, the empty LNP or loaded LNP comprises thestructural lipid in an amount from about 30% to about 50%. For example,in some embodiments, the empty LNP or loaded LNP comprises cholesterolin an amount from about 30% to about 50%.

In some embodiments, the empty LNP or loaded LNP comprises the PEG lipidin an amount from about 0% to about 5%. For example, in someembodiments, the empty LNP or loaded LNP comprises PEG-1 or PEG_(2k)-DMGin an amount from about 0% to about 5%.

In some embodiments, the empty LNP or loaded LNP comprises about 40 mol% to about 60 mol % of the compound of Formula (A), (A1), (A2), (A3),(B), (I), (I′), (I-X), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If),(Ig), (II), (IIa), or (IIb), about 0 mol % to about 20 mol %phospholipid, about 30 mol % to about 50 mol % structural lipid, andabout 0 mol % to about 5 mol % PEG lipid.

In some embodiments, the empty LNP or loaded LNP comprises about 40 mol% to about 60 mol % of the compound of Formula (A), (A1), (A2), (A3),(B), (I), (I′), (I-X), Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb), about 0 mol % to about 20 mol % DSPC,about 30 mol % to about 50 mol % cholesterol, and about 0 mol % to about5 mol % PEG_(2k)-DMG. In some embodiments, the empty LNP or loaded LNPcomprises about 40 mol % to about 60 mol % of the compound of Table 1,about 0 mol % to about 20 mol % DSPC, about 30 mol % to about 50 mol %cholesterol, and about 0 mol % to about 5 mol % PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises about 40 mol% to about 60 mol % of the compound of Formula (A), (A1), (A2), (A3),(B), (I), (I′), (I-X), (I-Y), Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), about 0 mol % to about 20 mol %DSPC, about 30 mol % to about 50 mol % cholesterol, and about 0 mol % toabout 5 mol % PEG-1. In some embodiments, the empty LNP or loaded LNPcomprises about 40 mol % to about 60 mol % of the compound of Table 1,about 0 mol % to about 20 mol % DSPC, about 30 mol % to about 50 mol %cholesterol, and about 0 mol % to about 5 mol % PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the structural lipid is cholesterol. In someembodiments, the empty LNP or loaded LNP comprises a compound of Table1, a phospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the structural lipid is cholesterol.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thestructural lipid is cholesterol and the PEG lipid is PEG_(2k)-DMG. Insome embodiments, the empty LNP or loaded LNP comprises a compound ofTable 1, a phospholipid, a structural lipid, and a PEG lipid, whereinthe structural lipid is cholesterol and the PEG lipid is PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thestructural lipid is cholesterol and the PEG lipid is PEG-1. In someembodiments, the empty LNP or loaded LNP comprises a compound of Table 1a phospholipid, a structural lipid, and a PEG lipid, wherein thestructural lipid is cholesterol and the PEG lipid is PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the PEG lipid is PEG_(2k)-DMG. In someembodiments, the empty LNP or loaded LNP comprises a compound of Table1, a phospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the PEG lipid is PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the PEG lipid is PEG-1. In some embodiments,the empty LNP or loaded LNP comprises a compound of Table 1, aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC and the PEG lipid is PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC, the structural lipid is cholesterol, and the PEGlipid is PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofTable 1, a phospholipid, a structural lipid, and a PEG lipid, whereinthe phospholipid is DSPC, the structural lipid is cholesterol, and thePEG lipid is PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), a phospholipid, a structural lipid, and a PEG lipid,wherein the phospholipid is DSPC, the structural lipid is cholesterol,and the PEG lipid is PEG_(2k)-DMG. In some embodiments, the empty LNP orloaded LNP comprises a compound of Table 1, a phospholipid, a structurallipid, and a PEG lipid, wherein the phospholipid is DSPC, the structurallipid is cholesterol, and the PEG lipid is PEG_(2k)-DMG.

In some embodiments, the empty LNP or loaded LNP comprises a compound of

Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, and a PEG lipid, wherein thephospholipid is DSPC, the structural lipid is cholesterol, and the PEGlipid is PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises a compound ofFormula (A), a phospholipid, a structural lipid, and a PEG lipid,wherein the phospholipid is DSPC, the structural lipid is cholesterol,and the PEG lipid is PEG-1. In some embodiments, the empty LNP or loadedLNP comprises a compound of Table 1, a phospholipid, a structural lipid,and a PEG lipid, wherein the phospholipid is DSPC, the structural lipidis cholesterol, and the PEG lipid is PEG-1.

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may be designedfor one or more specific applications or targets. For example, ananoparticle composition may be designed to deliver a therapeutic and/orprophylactic such as an RNA to a particular cell, tissue, organ, orsystem or group thereof in a mammal's body. Physiochemical properties oflipid nanoparticles (e.g., empty LNPs or loaded LNPs) may be altered inorder to increase selectivity for particular bodily targets. Forinstance, particle sizes may be adjusted based on the fenestration sizesof different organs. The therapeutic and/or prophylactic included in ananoparticle composition may also be selected based on the desireddelivery target or targets.

For example, a therapeutic and/or prophylactic may be selected for aparticular indication, condition, disease, or disorder and/or fordelivery to a particular cell, tissue, organ, or system or group thereof(e.g., localized or specific delivery). In certain embodiments, ananoparticle composition may include an mRNA encoding a polypeptide ofinterest capable of being translated within a cell to produce thepolypeptide of interest. Such a composition may be designed to bespecifically delivered to a particular organ. In some embodiments, acomposition may be designed to be specifically delivered to a mammalianliver.

The amount of a therapeutic and/or prophylactic in a nanoparticlecomposition may depend on the size, composition, desired target and/orapplication, or other properties of the nanoparticle composition as wellas on the properties of the therapeutic and/or prophylactic. Forexample, the amount of an RNA useful in a nanoparticle composition maydepend on the size, sequence, and other characteristics of the RNA. Therelative amounts of a therapeutic and/or prophylactic and other elements(e.g., lipids) in a nanoparticle composition may also vary. In someembodiments, the wt/wt ratio of the lipid component to a therapeuticand/or prophylactic in a nanoparticle composition may be from about 5:1to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1,50:1, and 60:1. For example, the wt/wt ratio of the lipid component to atherapeutic and/or prophylactic may be from about 10:1 to about 40:1. Incertain embodiments, the wt/wt ratio is about 20:1.

The amount of a therapeutic and/or prophylactic in a nanoparticlecomposition may, for example, be measured using absorption spectroscopy(e.g., ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition includes one or moreRNAs, and the one or more RNAs, lipids, and amounts thereof may beselected to provide a specific N:P ratio. The N:P ratio of thecomposition refers to the molar ratio of nitrogen atoms in one or morelipids to the number of phosphate groups in an RNA. In general, a lowerN:P ratio is preferred. The one or more RNA, lipids, and amounts thereofmay be selected to provide an N:P ratio from about 2:1 to about 30:1,such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1,18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, theN:P ratio may be from about 2:1 to about 8:1. In other embodiments, theN:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio maybe about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, orabout 7.0:1. For example, the N:P ratio may be about 5.67:1.

Physical Properties

The characteristics of a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may depend on the components thereof. For example, a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including cholesterolas a structural lipid may have different characteristics than a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) that includes adifferent structural lipid. Similarly, the characteristics of a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) may depend on theabsolute or relative amounts of its components. For instance, a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including a highermolar fraction of a phospholipid may have different characteristics thana lipid nanoparticle (e.g., an empty LNP or a loaded LNP) including alower molar fraction of a phospholipid. Characteristics may also varydepending on the method and conditions of preparation of thenanoparticle composition.

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may becharacterized by a variety of methods. For example, microscopy (e.g.,transmission electron microscopy or scanning electron microscopy) may beused to examine the morphology and size distribution of a nanoparticlecomposition. Dynamic light scattering or potentiometry (e.g.,potentiometric titrations) may be used to measure zeta potentials.Dynamic light scattering may also be utilized to determine particlesizes. Instruments such as the Zetasizer Nano ZS (Malvern InstrumentsLtd, Malvern, Worcestershire, UK) may also be used to measure multiplecharacteristics of a nanoparticle composition, such as particle size,polydispersity index, and zeta potential.

The mean size of a lipid nanoparticle (e.g., an empty LNP or a loadedLNP) may be between 10s of nm and 100s of nm, e.g., measured by dynamiclight scattering (DLS). For example, the mean size may be from about 40nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm,115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. Insome embodiments, the mean size of a lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) may be from about 50 nm to about 100 nm, from about50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nmto about 70 nm, from about 50 nm to about 60 nm, from about 60 nm toabout 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 150nm, from about 70 nm to about 130 nm, from about 70 nm to about 100 nm,from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, fromabout 80 nm to about 150 nm, from about 80 nm to about 130 nm, fromabout 80 nm to about 100 nm, from about 80 nm to about 90 nm, from about90 nm to about 150 nm, from about 90 nm to about 130 nm, or from about90 nm to about 100 nm. In certain embodiments, the mean size of a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) may from about 70 nmto about 130 nm or be from about 70 nm to about 100 nm. In a particularembodiment, the mean size may be about 80 nm. In other embodiments, themean size may be about 100 nm. In other embodiments, the mean size maybe about 120 nm.

A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may berelatively homogenous. A polydispersity index may be used to indicatethe homogeneity of a nanoparticle composition, e.g., the particle sizedistribution of the lipid nanoparticles (e.g., empty LNPs or loadedLNPs). A small (e.g., less than 0.3) polydispersity index generallyindicates a narrow particle size distribution. A lipid nanoparticle(e.g., an empty LNP or a loaded LNP) may have a polydispersity indexfrom about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18,0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, thepolydispersity index of a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may be from about 0.10 to about 0.20.

The zeta potential of a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may be used to indicate the electrokinetic potential of thecomposition. For example, the zeta potential may describe the surfacecharge of a nanoparticle composition. Lipid nanoparticles (e.g., emptyLNPs or loaded LNPs) with relatively low charges, positive or negative,are generally desirable, as more highly charged species may interactundesirably with cells, tissues, and other elements in the body. In someembodiments, the zeta potential of a lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) may be from about −10 mV to about +20 mV, fromabout −10 mV to about +15 mV, from about −10 mV to about +10 mV, fromabout −10 mV to about +5 mV, from about −10 mV to about 0 mV, from about−10 mV to about −5 mV, from about −5 mV to about +20 mV, from about −5mV to about +15 mV, from about −5 mV to about +10 mV, from about −5 mVto about +5 mV, from about −5 mV to about 0 mV, from about 0 mV to about+20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV,from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a therapeutic and/or prophylacticdescribes the amount of therapeutic and/or prophylactic that isencapsulated or otherwise associated with a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) after preparation, relative to the initialamount provided. The encapsulation efficiency is desirably high (e.g.,close to 100%). The encapsulation efficiency may be measured, forexample, by comparing the amount of therapeutic and/or prophylactic in asolution containing the lipid nanoparticle (e.g., an empty LNP or aloaded LNP) before and after breaking up the lipid nanoparticle (e.g.,an empty LNP or a loaded LNP) with one or more organic solvents ordetergents. Fluorescence may be used to measure the amount of freetherapeutic and/or prophylactic (e.g., RNA) in a solution. For the lipidnanoparticles (e.g., empty LNPs or loaded LNPs) described herein, theencapsulation efficiency of a therapeutic and/or prophylactic may be atleast 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments,the encapsulation efficiency may be at least 80%. In certainembodiments, the encapsulation efficiency may be at least 90%. In someembodiments, the encapsulation efficiency of the therapeutic and/orprophylactic agent is between 80% and 100%.

Pharmaceutical Compositions

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may be formulatedin whole or in part as pharmaceutical compositions. Pharmaceuticalcompositions may include one or more lipid nanoparticles (e.g., emptyLNPs or loaded LNPs). In one embodiment, a pharmaceutical compositioncomprises a population of lipid nanoparticles (e.g., empty LNPs orloaded LNPs). For example, a pharmaceutical composition may include oneor more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) includingone or more different therapeutic and/or prophylactics. Pharmaceuticalcompositions may further include one or more pharmaceutically acceptableexcipients or accessory ingredients such as those described herein.General guidelines for the formulation and manufacture of pharmaceuticalcompositions and agents are available, for example, in Remington's TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro;Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventionalexcipients and accessory ingredients may be used in any pharmaceuticalcomposition, except insofar as any conventional excipient or accessoryingredient may be incompatible with one or more components of ananoparticle composition. An excipient or accessory ingredient may beincompatible with a component of a lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) if its combination with the component may result inany undesirable biological effect or otherwise deleterious effect.

In some embodiments, one or more excipients or accessory ingredients maymake up greater than 50% of the total mass or volume of a pharmaceuticalcomposition including a nanoparticle composition. For example, the oneor more excipients or accessory ingredients may make up 50%, 60%, 70%,80%, 90%, or more of a pharmaceutical convention. In some embodiments, apharmaceutically acceptable excipient is at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% pure. In someembodiments, an excipient is approved for use in humans and forveterinary use. In some embodiments, an excipient is approved by UnitedStates Food and Drug Administration. In some embodiments, an excipientis pharmaceutical grade. In some embodiments, an excipient meets thestandards of the United States Pharmacopoeia (USP), the EuropeanPharmacopoeia (EP), the British Pharmacopoeia, and/or the InternationalPharmacopoeia.

Relative amounts of the one or more lipid nanoparticles (e.g., emptyLNPs or loaded LNPs), the one or more pharmaceutically acceptableexcipients, and/or any additional ingredients in a pharmaceuticalcomposition in accordance with the present disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, a pharmaceutical composition maycomprise between 0.1% and 100% (wt/wt) of one or more lipidnanoparticles (e.g., empty LNPs or loaded LNPs).

In certain embodiments, the lipid nanoparticles (e.g., empty LNPs orloaded LNPs) and/or pharmaceutical compositions of the disclosure arerefrigerated or frozen for storage and/or shipment (e.g., being storedat a temperature of 4° C. or lower, such as a temperature between about−150° C. and about 0° C. or between about −80° C. and about −20° C.(e.g., about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −40°C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C. or -150° C.).For example, the pharmaceutical composition comprising a compound of anyof Formulae (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb) isa solution that is refrigerated for storage and/or shipment at, forexample, about −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., or−80° C. In certain embodiments, the disclosure also relates to a methodof increasing stability of the lipid nanoparticles (e.g., empty LNPs orloaded LNPs) and/or pharmaceutical compositions comprising a compound ofany of Formulae (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A-3),(IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3), (A-b),(A-b1), (A-b2), (A-b3), (A-c), and (B-c) by storing the lipidnanoparticles (e.g., empty LNPs or loaded LNPs) and/or pharmaceuticalcompositions at a temperature of 4° C. or lower, such as a temperaturebetween about −150° C. and about 0° C. or between about −80° C. andabout −20° C., e.g., about −5° C., −10° C., −15° C., −20° C., −25° C.,−30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C.or -150° C.). For example, the lipid nanoparticles (e.g., empty LNPs orloaded LNPs) and/or pharmaceutical compositions disclosed herein arestable for about at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, atleast 2 months, at least 4 months, at least 6 months, at least 8 months,at least 10 months, at least 12 months, at least 14 months, at least 16months, at least 18 months, at least 20 months, at least 22 months, orat least 24 months, e.g., at a temperature of 4° C. or lower (e.g.,between about 4° C. and −20° C.). In some embodiments, the formulationis stabilized for at least 4 weeks at about 4° C. In certainembodiments, the pharmaceutical composition of the disclosure comprisesa lipid nanoparticle (e.g., an empty LNP or a loaded LNP) disclosedherein and a pharmaceutically acceptable carrier selected from one ormore of Tris, an acetate (e.g., sodium acetate), an citrate (e.g.,sodium citrate), saline, PBS, and sucrose. In certain embodiments, thepharmaceutical composition of the disclosure has a pH value betweenabout 7 and 8 (e.g., 6.8 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9 or 8.0, or between 7.5 and 8 or between 7 and 7.8). Forexample, a pharmaceutical composition of the disclosure comprises alipid nanoparticle (e.g., an empty LNP or a loaded LNP) disclosedherein, Tris, saline and sucrose, and has a pH of about 7.5-8, which issuitable for storage and/or shipment at, for example, about −20° C. Forexample, a pharmaceutical composition of the disclosure comprises alipid nanoparticle (e.g., an empty LNP or a loaded LNP) disclosed hereinand PBS and has a pH of about 7-7.8, suitable for storage and/orshipment at, for example, about 4° C. or lower. “Stability,”“stabilized,” and “stable” in the context of the present disclosurerefers to the resistance of lipid nanoparticles (e.g., empty LNPs orloaded LNPs) and/or pharmaceutical compositions disclosed herein tochemical or physical changes (e.g., degradation, particle size change,aggregation, change in encapsulation, etc.) under given manufacturing,preparation, transportation, storage and/or in-use conditions, e.g.,when stress is applied such as shear force, freeze/thaw stress, etc.

In some embodiments, a pharmaceutical composition of the disclosurecomprises a empty LNP or a loaded LNP, a cryoprotectant, a buffer, or acombination thereof.

In some embodiments, the cryoprotectant comprises one or morecryoprotective agents, and each of the one or more cryoprotective agentsis independently a polyol (e.g., a diol or a triol such as propyleneglycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol,(+/−)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol,2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergentsulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propane sulfonate), anosmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), apolymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG1000, PEG_(2k)-DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000,polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g.,polyvinylpyrrolidone K 15), pentaerythritol propoxylate, orpolypropylene glycol P 400), an organic solvent (e.g., dimethylsulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol,trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol,myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate,or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithiumchloride, lithium formate, lithium nitrate, lithium sulfate, magnesiumacetate, sodium acetate, sodium chloride, sodium formate, sodiummalonate, sodium nitrate, sodium sulfate, or any hydrate thereof), orany combination thereof. In some embodiments, the cryoprotectantcomprises sucrose. In some embodiments, the cryoprotectant and/orexcipient is sucrose. In some embodiments, the cryoprotectant comprisessodium acetate. In some embodiments, the cryoprotectant and/or excipientis sodium acetate. In some embodiments, the cryoprotectant comprisessucrose and sodium acetate.

In some embodiments, wherein the buffer is selected from the groupconsisting of an acetate buffer, a citrate buffer, a phosphate buffer, atris buffer, and combinations thereof.

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and/orpharmaceutical compositions including one or more lipid nanoparticles(e.g., empty LNPs or loaded LNPs) may be administered to any patient orsubject, including those patients or subjects that may benefit from atherapeutic effect provided by the delivery of a therapeutic and/orprophylactic to one or more particular cells, tissues, organs, orsystems or groups thereof. Although the descriptions provided herein oflipid nanoparticles (e.g., empty LNPs or loaded LNPs) and pharmaceuticalcompositions including lipid nanoparticles (e.g., empty LNPs or loadedLNPs) are principally directed to compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration to anyother mammal. Modification of compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation.

Subjects to which administration of the compositions is contemplatedinclude, but are not limited to, humans, other primates, and othermammals, including commercially relevant mammals such as cattle, pigs,hoses, sheep, cats, dogs, mice, and/or rats. The subject lipidnanoparticles can also be employed for in vitro and ex vivo uses.

A pharmaceutical composition including one or more lipid nanoparticles(e.g., empty LNPs or loaded LNPs) may be prepared by any method known orhereafter developed in the art of pharmacology. In general, suchpreparatory methods include bringing the active ingredient intoassociation with an excipient and/or one or more other accessoryingredients, and then, if desirable or necessary, dividing, shaping,and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient (e.g., nanoparticlecomposition). The amount of the active ingredient is generally equal tothe dosage of the active ingredient which would be administered to asubject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

Pharmaceutical compositions may be prepared in a variety of formssuitable for a variety of routes and methods of administration. Forexample, pharmaceutical compositions may be prepared in liquid dosageforms (e.g., emulsions, microemulsions, nanoemulsions, solutions,suspensions, syrups, and elixirs), injectable forms, solid dosage forms(e.g., capsules, tablets, pills, powders, and granules), dosage formsfor topical and/or transdermal administration (e.g., ointments, pastes,creams, lotions, gels, powders, solutions, sprays, inhalants, andpatches), suspensions, powders, and other forms.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, nanoemulsions, solutions, suspensions, syrups, and/orelixirs. In addition to active ingredients, liquid dosage forms maycomprise inert diluents commonly used in the art such as, for example,water or other solvents, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadditional therapeutic and/or prophylactics, additional agents such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and/or perfuming agents. In certain embodiments forparenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsulated matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofdrug to polymer and the nature of the particular polymer employed, therate of drug release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are prepared by entrapping the drug in liposomesor microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, films, powders, and granules. In such solid dosage forms, anactive ingredient is mixed with at least one inert, pharmaceuticallyacceptable excipient such as sodium citrate or dicalcium phosphateand/or fillers or extenders (e.g. starches, lactose, sucrose, glucose,mannitol, and silicic acid), binders (e.g., carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia),humectants (e.g., glycerol), disintegrating agents (e.g., agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate), solution retarding agents (e.g., paraffin),absorption accelerators (e.g., quaternary ammonium compounds), wettingagents (e.g., cetyl alcohol and glycerol monostearate), absorbents(e.g., kaolin and bentonite clay, silicates), and lubricants (e.g.,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate), and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants, and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices. Intradermalcompositions may be administered by devices which limit the effectivepenetration length of a needle into the skin.

Jet injection devices which deliver liquid compositions to the dermisvia a liquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable.Ballistic powder/particle delivery devices which use compressed gas toaccelerate vaccine in powder form through the outer layers of the skinto the dermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (wt/wt) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self-propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Drypowder compositions may include a solid fine powder diluent such assugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (wt/wt) of the composition, andactive ingredient may constitute 0.1% to 20% (wt/wt) of the composition.A propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 _(l)am to 500 _(l)am. Such a formulation is administered inthe manner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (wt/wt) and as much as 100%(wt/wt) of active ingredient, and may comprise one or more of theadditional ingredients described herein. A pharmaceutical compositionmay be prepared, packaged, and/or sold in a formulation suitable forbuccal administration. Such formulations may, for example, be in theform of tablets and/or lozenges made using conventional methods, andmay, for example, 0.1% to 20% (wt/wt) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (wt/wt) solution and/or suspension of the active ingredient inan aqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis present disclosure.

mRNA Therapies

mRNA as a drug modality has the potential to deliver secreted proteinsas well as intracellular proteins and transmembrane proteins. mRNA as adrug modality has the potential to deliver transmembrane andintracellular proteins, i.e., targets that standard biologics are unableto access owing to their inability to cross the cell membrane whendelivered in protein form. One major challenge to making mRNA basedtherapies a reality is the identification of an optimal deliveryvehicle. Due to its large size, chemical instability and potentialimmunogenicity, mRNA requires a delivery vehicle that can offerprotection from endo- and exo-nucleases, as well as shield the cargofrom immune sentinels. Lipid nanoparticles (LNPs) have been identifiedas a leading option in this regard.

Key performance criteria for a lipid nanoparticle delivery system are tomaximize cellular uptake and enable efficient release of mRNA from theendosome. In one embodiment, the subject LNPs comprising the novellipids disclosed herein, demonstrate improvements in at least one ofcellular uptake and endosomal release. At the same time the LNP mustprovide a stable drug product and be able to be dosed safely attherapeutically relevant levels. LNPs are multi-component systems whichtypically consist of an amino lipid, phospholipid, cholesterol, and aPEG-lipid. Each component is required for aspects of efficient deliveryof the nucleic acid cargo and stability of the particle. The keycomponent thought to drive cellular uptake, endosomal escape, andtolerability is the amino lipid. Cholesterol and the PEG-lipidcontribute to the stability of the drug product both in vivo and on theshelf, while the phospholipid provides additional fusogenicity to theLNP, thus helping to drive endosomal escape and rendering the nucleicacid bioavailable in the cytosol of cells.

Several amino lipid series have been developed for oligonucleotidedelivery over the past couple of decades, including the amino lipid MC3(DLin-MC3-DMA). MC3-based LNPs have been shown to be effective indelivering mRNA. LNPs of this class are quickly opsonized byapolipoprotein E (ApoE) when delivered intravenously, which enablescellular uptake by the low density lipoprotein receptor (LDLr). However,concerns remain that MC3′s long tissue half-life could contribute tounfavorable side effects hindering its use for chronic therapies. Inaddition, extensive literature evidence suggests that chronic dosing oflipid nanoparticles can produce several toxic sides effects includingcomplement activation-related pseudo allergy (CARPA) and liver damage.Hence, to unleash the potential of mRNA and other nucleic acid,nucleoptide or peptide based therapies for humans, a class of LNPs withincreased delivery efficiency along with a metabolic and toxicityprofile that would enable chronic dosing in humans is needed.

The ability to treat a broad swath of diseases requires the flexibilityto safely dose chronically at varying dose levels. Through systematicoptimization of the amino lipid structure, the compounds of thedisclosure were identified as compounds that balance chemical stability,improved efficiency of delivery due to improved endosomal escape, rapidin vivo metabolism, and a clean toxicity profile. The combination ofthese features provides a drug candidate that can be dosed chronicallywithout activation of the immune system. Initial rodent screens led tothe identification of a lead lipid with good delivery efficiency andpharmacokinetics. The lead LNP was profiled further in non-human primatefor efficiency of delivery after single and repeat dosing. Finally, theoptimized LNPs were evaluated in one-month repeat dose toxicity studiesin rat and non-human primate. Without wishing to be bound by theory, thenovel ionizable lipids of the instant disclosure have the improvedcellular delivery, improved protein expression, and improvedbiodegradability properties that can lead to greater than 2 fold, 5fold, 10 fold, 15 fold, or 20 fold increase in mRNA expression in cellsas compared to LNPs which lack a lipid of the invention. In anotherembodiment, an LNP comprising a lipid of the invention can result inspecific (e.g., preferential) delivery to a certain cell type or typesas compared other cell types, thereby resulting in a greater than 2fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in mRNA expressionin certain cells or tissues as compared to LNPs which lack a lipid ofthe invention. These improvements over the art allow for the safe andeffective use of mRNA-based therapies in acute and chronic diseases.

Methods

In some aspects, the disclosure provides a method of delivering atherapeutic and/or prophylactic to a cell (e.g., a mammalian cell). Thismethod includes the step of contacting the cell with a loaded LNP or apharmaceutical composition of the disclosure, whereby the therapeuticand/or prophylactic is delivered to the cell. In some embodiments, thecell is in a subject and the contacting comprises administering the cellto the subject. In some embodiments, the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, a PEG lipid, and one or moretherapeutic and/or prophylactic agents, whereby the therapeutic and/orprophylactic is delivered to the cell.

In some embodiments, the disclosure provides a method of delivering atherapeutic and/or prophylactic to a cell within a subject, wherein themethod comprises the step of administering to the subject a lipidnanoparticle comprising a compound of Formula (A), (A1), (A2), (A3),(B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, and PEG_(2k)-DMG,and one or more therapeutic and/or prophylactic agents selected from anucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). Forexample, in some embodiments, the disclosure provides a method ofdelivering a therapeutic and/or prophylactic to a cell within a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA).

In some embodiments, the disclosure provides a method of delivering atherapeutic and/or prophylactic to a cell within a subject, wherein themethod comprises the step of administering to the subject a lipidnanoparticle comprising a compound of Formula (A), (A1), (A2), (A3),(B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, and PEG-1, and oneor more therapeutic and/or prophylactic agents selected from anucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). Forexample, in some embodiments, the disclosure provides a method ofdelivering a therapeutic and/or prophylactic to a cell within a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG-1, and one or more therapeutic and/or prophylacticagents selected from a nucleotide, a polypeptide, and a nucleic acid(e.g., an RNA).

In some aspects, the disclosure provides a method of delivering (e.g.,specifically delivering) a therapeutic and/or prophylactic to amammalian organ or tissue (e.g., a liver, kidney, spleen, or lung). Thismethod includes the step of contacting the cell with a loaded LNP or apharmaceutical composition of the disclosure, whereby the therapeuticand/or prophylactic is delivered to the target organ or tissue. In someembodiments, the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Formula (A), (A1),(A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic),(Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), a phospholipid, astructural lipid, a PEG lipid, and one or more therapeutic and/orprophylactic agents, whereby the therapeutic and/or prophylactic isdelivered to the target organ or tissue.

In some embodiments, the disclosure provides a method of specificallydelivering a therapeutic and/or prophylactic to an organ of a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, andPEG_(2k)-DMG, and one or more therapeutic and/or prophylactic agentsselected from a nucleotide, a polypeptide, and a nucleic acid (e.g., anRNA). For example, in some embodiments, the disclosure provides a methodof specifically delivering a therapeutic and/or prophylactic to an organof a subject, wherein the method comprises the step of administering tothe subject a lipid nanoparticle comprising a compound of Formula (A),DSPC, cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA).

In some embodiments, the disclosure provides a method of specificallydelivering a therapeutic and/or prophylactic to an organ of a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, and PEG-1,and one or more therapeutic and/or prophylactic agents selected from anucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). Forexample, in some embodiments, the disclosure provides a method ofspecifically delivering a therapeutic and/or prophylactic to an organ ofa subject, wherein the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG-1, and one or more therapeutic and/or prophylacticagents selected from a nucleotide, a polypeptide, and a nucleic acid(e.g., an RNA).

In some aspects, the disclosure features a method for the enhanceddelivery of a therapeutic and/or prophylactic (e.g., an mRNA) to atarget tissue (e.g., a liver, spleen, or lung). This method includes thestep of contacting the cell with a loaded LNP or a pharmaceuticalcomposition of the disclosure, whereby the therapeutic and/orprophylactic is delivered to the target tissue (e.g., a liver, kidney,spleen, or lung). In some embodiments, the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, a PEG lipid, and one or moretherapeutic and/or prophylactic agents, whereby the therapeutic and/orprophylactic is delivered to the target tissue (e.g., a liver, kidney,spleen, or lung).

In some embodiments, the disclosure provides a method for the enhanceddelivery of a therapeutic and/or prophylactic to a target tissue,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, andPEG_(2k)-DMG, and one or more therapeutic and/or prophylactic agentsselected from a nucleotide, a polypeptide, and a nucleic acid (e.g., anRNA). For example, in some embodiments, the disclosure provides a methodfor the enhanced delivery of a therapeutic and/or prophylactic to atarget tissue, wherein the method comprises the step of administering tothe subject a lipid nanoparticle comprising a compound of Formula (A),DSPC, cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA).

In some embodiments, the disclosure provides a method for the enhanceddelivery of a therapeutic and/or prophylactic to a target tissue,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), DSPC, cholesterol, and PEG-1,and one or more therapeutic and/or prophylactic agents selected from anucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). Forexample, in some embodiments, the disclosure provides a method for theenhanced delivery of a therapeutic and/or prophylactic to a targettissue, wherein the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG-1, and one or more therapeutic and/or prophylacticagents selected from a nucleotide, a polypeptide, and a nucleic acid(e.g., an RNA).

In some aspects, the disclosure provides a method of producing apolypeptide of interest in a cell (e.g., a mammalian cell). This methodincludes the step of contacting the cell with a loaded LNP or apharmaceutical composition of the disclosure, wherein the loaded LNP orpharmaceutical composition comprises an mRNA, whereby the mRNA iscapable of being translated in the cell to produce the polypeptide. Insome embodiments, the cell is in a subject and the contacting comprisesadministering the cell to the subject. In some embodiments, the methodcomprises the step of administering to the subject a lipid nanoparticlecomprising a compound of Formula (A), (A1), (A2), (A3), (B), (I), (I′),(I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),(II), (IIa), or (IIb), a phospholipid, a structural lipid, a PEG lipid,and an mRNA, whereby the mRNA is capable of being translated in the cellto produce the polypeptide.

In some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), DSPC, cholesterol, and PEG_(2k)-DMG, and an mRNA. For example, insome embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of Table 1, DSPC, cholesterol, and PEG_(2k)-DMG, and an mRNA.For example, in some embodiments, the disclosure provides a method ofproducing a polypeptide of interest in a cell, wherein the methodcomprises the step of administering to the subject a lipid nanoparticlecomprising a compound of Formula (A), DSPC, cholesterol, andPEG_(2k)-DMG, and an mRNA.

In some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),DSPC, cholesterol, and PEG-1, and an mRNA. For example, in someembodiments, the disclosure provides a method of producing a polypeptideof interest in a cell, wherein the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), DSPC, cholesterol, and PEG-1, and an mRNA. For example,in some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of Table 1, DSPC, cholesterol, and PEG-1, and an mRNA.

In some aspects, the disclosure provides a method of treating a diseaseor disorder in a mammal (e.g., a human) in need thereof. The methodincludes the step of administering to the mammal a therapeuticallyeffective amount of loaded LNP or a pharmaceutical composition of thedisclosure. In some embodiments, the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, a PEG lipid, and one or moretherapeutic and/or prophylactic agents, whereby the therapeutic and/orprophylactic is delivered to the cell. In some embodiments, the diseaseor disorder is characterized by dysfunctional or aberrant protein orpolypeptide activity. For example, the disease or disorder is selectedfrom the group consisting of rare diseases, infectious diseases, cancerand proliferative diseases, genetic diseases, autoimmune diseases,diabetes, neurodegenerative diseases, cardio- and reno-vasculardiseases, and metabolic diseases.

In some embodiments, the disclosure provides a method of treating adisease or disorder in a subject, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), DSPC, cholesterol, and PEG_(2k)-DMG, and one or more therapeuticand/or prophylactic agents selected from a nucleotide, a polypeptide,and a nucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of treating a disease or disorder in asubject, wherein the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of treating a disease or disorder in asubject, wherein the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Table 1, DSPC,cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA).

In some embodiments, the disclosure provides a method of treating adisease or disorder in a subject, wherein the method comprises the stepof administering to the subject a lipid nanoparticle comprising acompound of Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y),(I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or(IIb), DSPC, cholesterol, and PEG-1, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of treating a disease or disorder in asubject, wherein the method comprises the step of administering to thesubject a lipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG-1, and one or more therapeutic and/or prophylacticagents selected from a nucleotide, a polypeptide, and a nucleic acid(e.g., an RNA). For example, in some embodiments, the disclosureprovides a method of treating a disease or disorder in a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol,and PEG-1, and one or more therapeutic and/or prophylactic agentsselected from a nucleotide, a polypeptide, and a nucleic acid (e.g., anRNA).

In yet another aspect, the disclosure features a method of loweringimmunogenicity comprising introducing loaded LNP or a pharmaceuticalcomposition of the disclosure into cells, wherein the loaded LNP or apharmaceutical composition reduces the induction of the cellular immuneresponse of the cells to the loaded LNP or a pharmaceutical composition,as compared to the induction of the cellular immune response in cellsinduced by a reference composition. In some embodiments, the cell is ina subject and the contacting comprises administering the cell to thesubject. In some embodiments, the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb), aphospholipid, a structural lipid, a PEG lipid, and one or moretherapeutic and/or prophylactic agents selected from a nucleotide, apolypeptide, and a nucleic acid (e.g., an RNA), wherein the lipidnanoparticle comprising a compound of

Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb)reduces the induction of the cellular immune response of the cells tothe lipid nanoparticle comprising a compound of Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), as compared to the induction ofthe cellular immune response in cells induced by a referencecomposition. For example, the cellular immune response is an innateimmune response, an adaptive immune response, or both.

In some embodiments, the disclosure provides a method of loweringimmunogenicity in a subject, wherein the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),DSPC, cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of lowering immunogenicity in a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG_(2k)-DMG, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of lowering immunogenicity in a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Table 1, DSPC, cholesterol,and PEG_(2k)-DMG, and one or more therapeutic and/or prophylactic agentsselected from a nucleotide, a polypeptide, and a nucleic acid (e.g., anRNA).

In some embodiments, the disclosure provides a method of loweringimmunogenicity in a subject, wherein the method comprises the step ofadministering to the subject a lipid nanoparticle comprising a compoundof Formula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),DSPC, cholesterol, and PEG-1, and one or more therapeutic and/orprophylactic agents selected from a nucleotide, a polypeptide, and anucleic acid (e.g., an RNA). For example, in some embodiments, thedisclosure provides a method of lowering immunogenicity in a subject,wherein the method comprises the step of administering to the subject alipid nanoparticle comprising a compound of Formula (A), DSPC,cholesterol, and PEG-1, and one or more therapeutic and/or prophylacticagents selected from a nucleotide, a polypeptide, and a nucleic acid(e.g., an RNA). For example, in some embodiments, the disclosureprovides a method of lowering immunogenicity in a subject, wherein themethod comprises the step of administering to the subject a lipidnanoparticle comprising a compound of Table 1, DSPC, cholesterol, andPEG-1, and one or more therapeutic and/or prophylactic agents selectedfrom a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

The disclosure also includes methods of synthesizing a compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),and methods of making a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) including a lipid component comprising the compound ofFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb).

Methods of Producing Polypeptides in Cells

The present disclosure provides methods of producing a polypeptide ofinterest in a mammalian cell. Methods of producing polypeptides involvecontacting a cell with a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) including an mRNA encoding the polypeptide of interest. Uponcontacting the cell with the nanoparticle composition, the mRNA may betaken up and translated in the cell to produce the polypeptide ofinterest.

In general, the step of contacting a mammalian cell with a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including an mRNAencoding a polypeptide of interest may be performed in vivo, ex vivo, inculture, or in vitro. The amount of lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) contacted with a cell, and/or the amount of mRNAtherein, may depend on the type of cell or tissue being contacted, themeans of administration, the physiochemical characteristics of the lipidnanoparticle (e.g., an empty LNP or a loaded LNP) and the mRNA (e.g.,size, charge, and chemical composition) therein, and other factors. Ingeneral, an effective amount of the lipid nanoparticle (e.g., an emptyLNP or a loaded LNP) will allow for efficient polypeptide production inthe cell. Metrics for efficiency may include polypeptide translation(indicated by polypeptide expression), level of mRNA degradation, andimmune response indicators.

The step of contacting a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) including an mRNA with a cell may involve or causetransfection. A phospholipid including in the lipid component of a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) may facilitatetransfection and/or increase transfection efficiency, for example, byinteracting and/or fusing with a cellular or intracellular membrane.Transfection may allow for the translation of the mRNA within the cell.

In some embodiments, the lipid nanoparticles (e.g., empty LNPs or loadedLNPs) described herein may be used therapeutically. For example, an mRNAincluded in a lipid nanoparticle (e.g., an empty LNP or a loaded LNP)may encode a therapeutic polypeptide (e.g., in a translatable region)and produce the therapeutic polypeptide upon contacting and/or entry(e.g., transfection) into a cell. In other embodiments, an mRNA includedin a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may encodea polypeptide that may improve or increase the immunity of a subject.For example, an mRNA may encode a granulocyte-colony stimulating factoror trastuzumab.

In certain embodiments, an mRNA included in a lipid nanoparticle (e.g.,an empty LNP or a loaded LNP) may encode a recombinant polypeptide thatmay replace one or more polypeptides that may be substantially absent ina cell contacted with the nanoparticle composition. The one or moresubstantially absent polypeptides may be lacking due to a geneticmutation of the encoding gene or a regulatory pathway thereof.Alternatively, a recombinant polypeptide produced by translation of themRNA may antagonize the activity of an endogenous protein present in, onthe surface of, or secreted from the cell. An antagonistic recombinantpolypeptide may be desirable to combat deleterious effects caused byactivities of the endogenous protein, such as altered activities orlocalization caused by mutation. In another alternative, a recombinantpolypeptide produced by translation of the mRNA may indirectly ordirectly antagonize the activity of a biological moiety present in, onthe surface of, or secreted from the cell. Antagonized biologicalmoieties may include, but are not limited to, lipids (e.g.,cholesterol), lipoproteins (e.g., low density lipoprotein), nucleicacids, carbohydrates, and small molecule toxins. Recombinantpolypeptides produced by translation of the mRNA may be engineered forlocalization within the cell, such as within a specific compartment suchas the nucleus, or may be engineered for secretion from the cell or fortranslocation to the plasma membrane of the cell.

In some embodiments, contacting a cell with a lipid nanoparticle (e.g.,an empty LNP or a loaded LNP) including an mRNA may reduce the innateimmune response of a cell to an exogenous nucleic acid. A cell may becontacted with a first lipid nanoparticle (e.g., an empty LNP or aloaded LNP) including a first amount of a first exogenous mRNA includinga translatable region and the level of the innate immune response of thecell to the first exogenous mRNA may be determined. Subsequently, thecell may be contacted with a second composition including a secondamount of the first exogenous mRNA, the second amount being a lesseramount of the first exogenous mRNA compared to the first amount.Alternatively, the second composition may include a first amount of asecond exogenous mRNA that is different from the first exogenous mRNA.The steps of contacting the cell with the first and second compositionsmay be repeated one or more times. Additionally, efficiency ofpolypeptide production (e.g., translation) in the cell may be optionallydetermined, and the cell may be re-contacted with the first and/orsecond composition repeatedly until a target protein productionefficiency is achieved.

Methods of Delivering Therapeutic Agents to Cells and Organs

The present disclosure provides methods of delivering a therapeuticand/or prophylactic to a mammalian cell or organ. Delivery of atherapeutic and/or prophylactic to a cell involves administering a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including thetherapeutic and/or prophylactic to a subject, where administration ofthe composition involves contacting the cell with the composition. Forexample, a protein, cytotoxic agent, radioactive ion, chemotherapeuticagent, or nucleic acid (such as an RNA, e.g., mRNA) may be delivered toa cell or organ. In the instance that a therapeutic and/or prophylacticis an mRNA, upon contacting a cell with the nanoparticle composition, atranslatable mRNA may be translated in the cell to produce a polypeptideof interest. However, mRNAs that are substantially not translatable mayalso be delivered to cells.

Substantially non-translatable mRNAs may be useful as vaccines and/ormay sequester translational components of a cell to reduce expression ofother species in the cell.

In some embodiments, a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may target a particular type or class of cells (e.g., cellsof a particular organ or system thereof). For example, a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including atherapeutic and/or prophylactic of interest may be specificallydelivered to a mammalian liver, kidney, spleen, or lung. Specificdelivery to a particular class of cells, an organ, or a system or groupthereof implies that a higher proportion of lipid nanoparticles (e.g.,loaded LNPs) including a therapeutic and/or prophylactic are deliveredto the destination (e.g., tissue) of interest relative to otherdestinations. In some embodiments, specific delivery of a loaded LNPcomprising an mRNA may result in a greater than 2 fold, 5 fold, 10 fold,15 fold, or 20 fold increase in mRNA expression in cells of the targeteddestination (e.g., tissue of interest, such as a liver) as compared tocells of another destination (e.g., the spleen). In some embodiments,the tissue of interest is selected from the group consisting of a liver,a kidney, a lung, a spleen, and tumor tissue (e.g., via intratumoralinjection).

In some embodiments, specific delivery of an mRNA comprised in a loadedLNP of the disclosure (i.e., a lipid nanoparticle formulated with acompound of the disclosure) may result in a greater than 2 fold, 5 fold,10 fold, 15 fold, or 20 fold increase in mRNA expression as compared todelivery of an mRNA comprised in an LNP formulated with another lipid(i.e., without any of the lipids of Formula (A), (A1), (A2), (A3), (B),(I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (II), (IIa), or (IIb).

As another example of targeted or specific delivery, an mRNA thatencodes a protein-binding partner (e.g., an antibody or functionalfragment thereof, a scaffold protein, or a peptide) or a receptor on acell surface may be included in a nanoparticle composition. An mRNA mayadditionally or instead be used to direct the synthesis andextracellular localization of lipids, carbohydrates, or other biologicalmoieties. Alternatively, other therapeutic and/or prophylactics orelements (e.g., lipids or ligands) of a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) may be selected based on their affinity forparticular receptors (e.g., low density lipoprotein receptors) such thata lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may morereadily interact with a target cell population including the receptors.For example, ligands may include, but are not limited to, members of aspecific binding pair, antibodies, monoclonal antibodies, Fv fragments,single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2 fragments,single domain antibodies, camelized antibodies and fragments thereof,humanized antibodies and fragments thereof, and multivalent versionsthereof; multivalent binding reagents including mono- or bi-specificantibodies such as disulfide stabilized Fv fragments, scFv tandems,diabodies, tribodies, or tetrabodies; and aptamers, receptors, andfusion proteins.

In some embodiments, a ligand may be a surface-bound antibody, which canpermit tuning of cell targeting specificity. This is especially usefulsince highly specific antibodies can be raised against an epitope ofinterest for the desired targeting site. In some embodiments, multipleantibodies are expressed on the surface of a cell, and each antibody canhave a different specificity for a desired target. Such approaches canincrease the avidity and specificity of targeting interactions.

A ligand can be selected, e.g., by a person skilled in the biologicalarts, based on the desired localization or function of the cell.

Targeted cells may include, but are not limited to, hepatocytes,epithelial cells, hematopoietic cells, epithelial cells, endothelialcells, lung cells, bone cells, stem cells, mesenchymal cells, neuralcells, cardiac cells, adipocytes, vascular smooth muscle cells,cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells,synovial lining cells, ovarian cells, testicular cells, fibroblasts, Bcells, T cells, reticulocytes, leukocytes, granulocytes, and tumorcells.

In some embodiments, a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may target hepatocytes. Apolipoprotiens such asapolipoprotein E (apoE) have been shown to associate with neutral ornear neutral lipid-containing lipid nanoparticles (e.g., empty LNPs orloaded LNPs) in the body, and are known to associate with receptors suchas low-density lipoprotein receptors (LDLRs) found on the surface ofhepatocytes. Thus, a lipid nanoparticle (e.g., an empty LNP or a loadedLNP) including a lipid component with a neutral or near neutral chargethat is administered to a subject may acquire apoE in a subject's bodyand may subsequently deliver a therapeutic and/or prophylactic (e.g., anRNA) to hepatocytes including LDLRs in a targeted manner.

Methods of Treating Diseases and Disorders

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may be useful fortreating a disease, disorder, or condition. In particular, suchcompositions may be useful in treating a disease, disorder, or conditioncharacterized by missing or aberrant protein or polypeptide activity.For example, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP)comprising an mRNA encoding a missing or aberrant polypeptide may beadministered or delivered to a cell. Subsequent translation of the mRNAmay produce the polypeptide, thereby reducing or eliminating an issuecaused by the absence of or aberrant activity caused by the polypeptide.Because translation may occur rapidly, the methods and compositions maybe useful in the treatment of acute diseases, disorders, or conditionssuch as sepsis, stroke, and myocardial infarction. A therapeutic and/orprophylactic included in a lipid nanoparticle (e.g., an empty LNP or aloaded LNP) may also be capable of altering the rate of transcription ofa given species, thereby affecting gene expression.

Diseases, disorders, and/or conditions characterized by dysfunctional oraberrant protein or polypeptide activity for which a composition may beadministered include, but are not limited to, rare diseases, infectiousdiseases (as both vaccines and therapeutics), cancer and proliferativediseases, genetic diseases, autoimmune diseases, diabetes,neurodegenerative diseases, cardio- and reno-vascular diseases, andmetabolic diseases. Multiple diseases, disorders, and/or conditions maybe characterized by missing (or substantially diminished such thatproper protein function does not occur) protein activity. Such proteinsmay not be present, or they may be essentially non-functional. Thepresent disclosure provides a method for treating such diseases,disorders, and/or conditions in a subject by administering a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) including an RNA and alipid component including a lipid according to Formula (A), (A1), (A2),(A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (II), (IIa), or (IIb), a phospholipid (optionallyunsaturated), a PEG lipid, and a structural lipid, wherein the RNA maybe an mRNA encoding a polypeptide that antagonizes or otherwiseovercomes an aberrant protein activity present in the cell of thesubject.

The disclosure provides methods involving administering lipidnanoparticles (e.g., empty LNPs or loaded LNPs) including one or moretherapeutic and/or prophylactic agents and pharmaceutical compositionsincluding the same. The terms therapeutic and prophylactic can be usedinterchangeably herein with respect to features and embodiments of thepresent disclosure. Therapeutic compositions, or imaging, diagnostic, orprophylactic compositions thereof, may be administered to a subjectusing any reasonable amount and any route of administration effectivefor preventing, treating, diagnosing, or imaging a disease, disorder,and/or condition and/or any other purpose. The specific amountadministered to a given subject may vary depending on the species, age,and general condition of the subject; the purpose of the administration;the particular composition; the mode of administration; and the like.Compositions in accordance with the present disclosure may be formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage of acomposition of the present disclosure will be decided by an attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or otherwiseappropriate dose level (e.g., for imaging) for any particular patientwill depend upon a variety of factors including the severity andidentify of a disorder being treated, if any; the one or moretherapeutic and/or prophylactics employed; the specific compositionemployed; the age, body weight, general health, sex, and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific pharmaceutical composition employed; theduration of the treatment; drugs used in combination or coincidentalwith the specific pharmaceutical composition employed; and like factorswell known in the medical arts.

A loaded LNP may be administered by any route. In some embodiments,compositions, including prophylactic, diagnostic, or imagingcompositions including one or more loaded LNPs described herein, areadministered by one or more of a variety of routes, including oral,intravenous, intramuscular, intra-arterial, subcutaneous, trans- orintra-dermal, interdermal, intraperitoneal, mucosal, nasal,intratumoral, intranasal; by inhalation; as an oral spray and/or powder,nasal spray, and/or aerosol, and/or through a portal vein catheter. Insome embodiments, a composition may be administered intravenously,intramuscularly, intradermally, intra-arterially, intratumorally,subcutaneously, or by any other parenteral route of administration or byinhalation. However, the present disclosure encompasses the delivery oradministration of compositions described herein by any appropriate routetaking into consideration likely advances in the sciences of drugdelivery. In general, the most appropriate route of administration willdepend upon a variety of factors including the nature of the loaded LNPincluding one or more therapeutic and/or prophylactics (e.g., itsstability in various bodily environments such as the bloodstream andgastrointestinal tract), the condition of the patient (e.g., whether thepatient is able to tolerate particular routes of administration), etc.

In certain embodiments, compositions in accordance with the presentdisclosure may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg toabout 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 10 mg/kg,from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, fromabout 0.01 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 5mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 1 mg/kg toabout 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 0.0001mg/kg to about 2.5 mg/kg, from about 0.001 mg/kg to about 2.5 mg/kg,from about 0.005 mg/kg to about 2.5 mg/kg, from about 0.01 mg/kg toabout 2.5 mg/kg, from about 0.05 mg/kg to about 2.5 mg/kg, from about0.1 mg/kg to about 2.5 mg/kg, from about 1 mg/kg to about 2.5 mg/kg,from about 2 mg/kg to about 2.5 mg/kg, from about 0.0001 mg/kg to about1 mg/kg, from about 0.001 mg/kg to about 1 mg/kg, from about 0.005 mg/kgto about 1 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, from about0.05 mg/kg to about 1 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, fromabout 0.0001 mg/kg to about 0.25 mg/kg, from about 0.001 mg/kg to about0.25 mg/kg, from about 0.005 mg/kg to about 0.25 mg/kg, from about 0.01mg/kg to about 0.25 mg/kg, from about 0.05 mg/kg to about 0.25 mg/kg, orfrom about 0.1 mg/kg to about 0.25 mg/kg of a therapeutic and/orprophylactic (e.g., an mRNA) in a given dose, where a dose of 1 mg/kg(mpk) provides 1 mg of a therapeutic and/or prophylactic per 1 kg ofsubject body weight. In some embodiments, a dose of about 0.001 mg/kg toabout 10 mg/kg of a therapeutic and/or prophylactic of a loaded LNP maybe administered. In other embodiments, a dose of about 0.005 mg/kg toabout 2.5 mg/kg of a therapeutic and/or prophylactic may beadministered. In certain embodiments, a dose of about 0.1 mg/kg to about1 mg/kg may be administered. In other embodiments, a dose of about 0.05mg/kg to about 0.25 mg/kg may be administered. A dose may beadministered one or more times per day, in the same or a differentamount, to obtain a desired level of mRNA expression and/or therapeutic,diagnostic, prophylactic, or imaging effect. The desired dosage may bedelivered, for example, three times a day, two times a day, once a day,every other day, every third day, every week, every two weeks, everythree weeks, or every four weeks. In certain embodiments, the desireddosage may be delivered using multiple administrations (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations). In some embodiments, asingle dose may be administered, for example, prior to or after asurgical procedure or in the instance of an acute disease, disorder, orcondition.

Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) including one ormore therapeutic and/or prophylactics may be used in combination withone or more other therapeutic, prophylactic, diagnostic, or imagingagents. By “in combination with,” it is not intended to imply that theagents must be administered at the same time and/or formulated fordelivery together, although these methods of delivery are within thescope of the present disclosure. For example, one or more lipidnanoparticles (e.g., empty LNPs or loaded LNPs) including one or moredifferent therapeutic and/or prophylactics may be administered incombination. Compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thepresent disclosure encompasses the delivery of compositions, or imaging,diagnostic, or prophylactic compositions thereof in combination withagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination will be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination may be lower than those utilizedindividually.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer may be administeredconcurrently with a chemotherapeutic agent), or they may achievedifferent effects (e.g., control of any adverse effects, such asinfusion related reactions).

A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may be used incombination with an agent to increase the effectiveness and/ortherapeutic window of the composition. Such an agent may be, forexample, an anti-inflammatory compound, a steroid (e.g., acorticosteroid), a statin, an estradiol, a BTK inhibitor, an S1P1agonist, a glucocorticoid receptor modulator (GRM), or ananti-histamine. In some embodiments, a lipid nanoparticle (e.g., anempty LNP or a loaded LNP) may be used in combination withdexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, oran H2 receptor blocker. In some embodiments, a method of treating asubject in need thereof or of delivering a therapeutic and/orprophylactic to a subject (e.g., a mammal) may involve pre-treating thesubject with one or more agents prior to administering a nanoparticlecomposition. For example, a subject may be pre-treated with a usefulamount (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90mg, 100 mg, or any other useful amount) of dexamethasone, methotrexate,acetaminophen, an H1 receptor blocker, or an H2 receptor blocker.Pre-treatment may occur 24 or fewer hours (e.g., 24 hours, 20 hours, 16hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40minutes, 30 minutes, 20 minutes, or 10 minutes) before administration ofthe lipid nanoparticle (e.g., an empty LNP or a loaded LNP) and mayoccur one, two, or more times in, for example, increasing dosageamounts.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all, of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. As used herein, the expressions “one or moreof A, B, or C,” “one or more A, B, or C,” “one or more of A, B, and C,”“one or more A, B, and C”, “selected from A, B, and C,” “selected fromthe group consisting of A, B, and C,” and the like are usedinterchangeably and all refer to a selection from a group consisting ofA, B, and/or C, i.e., one or more As, one or more Bs, one or more Cs, orany combination thereof, unless otherwise specified.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the terms “consistingessentially of” and “consisting of” are thus also encompassed anddisclosed. Throughout the description, where compositions are describedas having, including, or comprising specific components, it iscontemplated that compositions also consist essentially of, or consistof, the recited components. Similarly, where methods or processes aredescribed as having, including, or comprising specific process steps,the processes also consist essentially of, or consist of, the recitedprocessing steps. Further, it should be understood that the order ofsteps or order for performing certain actions is immaterial so long asthe invention remains operable. Moreover, two or more steps or actionscan be conducted simultaneously.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt thereof.

Compounds of the present disclosure can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001;Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999; R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentdisclosure.

The compounds of this disclosure having any of the formulae describedherein may be prepared according to the procedures illustrated inSchemes 1, 2, and 3 below, from commercially available startingmaterials or starting materials which can be prepared using literatureprocedures. The variables in the schemes (e.g., R¹, R², and R³ etc. areas defined herein). One of ordinary skill in the art will note that,during the reaction sequences and synthetic schemes described herein,the order of certain steps may be changed, such as the introduction andremoval of protecting groups.

One of ordinary skill in the art will recognize that certain groups mayrequire protection from the reaction conditions via the use ofprotecting groups. Protecting groups may also be used to differentiatesimilar functional groups in molecules. A list of protecting groups andhow to introduce and remove these groups can be found in Greene, T. W.,Wuts, P.G. M., Protective Groups in Organic Synthesis, 3^(rd) edition,John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For a hydroxyl moiety: TBS, benzyl, THP, Ac;

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allylester;

For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl,phthalimide, benzylideneamine;

For diols: Ac (x2) TBS (x2), or when taken together acetonides;

For thiols: Ac;

For benzimidazoles: SEM, benzyl, PMB, DMB;

For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

As illustrated in Scheme 1 above, 8-bromooctanoic acid reacts with analcohol a1 (e.g., heptadecan-9-ol) to afford an ester b1 (e.g.,heptadecan-9-yl 8-bromooctanoate). Step 1 can take place in an organicsolvent (e.g., dichloromethane) in the presence of, e.g.,N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride,N,N-diisopropylethylamine and DMAP. Step 1 can take place at roomtemperature for 18 h. Next, ester b1 reacts with 2-aminoethan-1-ol toafford amine cl (e.g., heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate). Step 2 can take place in ethanolat, e.g., a temperature of about 60° C. Then amine c1 reacts with anbromoalkyl R¹-Br (e.g., 1-bromotetradecane) to afford compound d1 (e.g.,heptadecan-9-yl 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate). Step 3can take place in ethanol in the presence of N,N-diisopropylethylamine.

As illustrated in Scheme 2 above, an acid a2 (x³ is an integer between 1and 7; e.g., 8-bromooctanoic acid) reacts with an alcohol b2 (e.g.,nonan-1-ol) to afford an ester c2 (e.g., nonyl-8-bromooctanoate). Step 1can take place in an organic solvent (e.g., dichloromethane) in thepresence of, e.g., N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride, N,N-diisopropylethylamine and DMAP. Alcohol e2 (e.g.,heptadecan-9-ol) can be obtained from reacting aldehyde d2 (e.g.,nonanal) with a Grignard reagent R³-MgX (e.g., n-C₈H₁₇MgBr) via Step 2.Next, 8-bromooctanoic acid reacts with an alcohol e2 (e.g.,heptadecan-9-ol) to afford an ester f2 (e.g., heptadecan-9-yl8-bromooctanoate). Step 3 can take place in an organic solvent (e.g.,dichloromethane) in the presence of, e.g.,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,N,N-diisopropylethylamine and DMAP. Next, ester f2 reacts with2-aminoethan-1-ol to afford amine g2 (e.g., heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate). Step 4 can take place in ethanol inthe presence of i-Pr2EtN. Then amine g2 reacts with ester c2 (e.g.,nonyl-8-bromooctanoate) to afford compound h2 (e.g., heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate). Step 5 cantake place in an organic solvent (e.g., a mixture of CPME and MeCN), inthe presence of a base (such as an inorganic base (e.g., K₂CO₃) ornon-nucleophilic organic base (e.g., i-Pr₂EtN)) and a catalyst (e.g., aniodide such as KI or NaI) at, e.g., an elevated temperature (such as atabout 70-90° C., e.g., about 80° C.).

As illustrated in Scheme 3 above, a haloalkanol (x³ is an integerbetween 1 and 12, e.g., 6-bromohexan-1-ol) is reacted with a startingmaterial a3 (x² is an integer between 1 and 6, e.g.,4-(hexyloxy)-4-oxobutanoic acid) to afford a halogenated diester b3(e.g., 6-bromohexyl hexyl succinate). Compound a3 can be obtained byreaction of an alcohol (e.g., hexan-1-ol) with an acid anhydride (e.g.succinic anhydride, dihydro-2H-pyran-2,6(3H)-dione,3-(tert-butoxy)-3-oxopropanoic acid,4-(tert-butoxy)-3-methyl-4-oxobutanoic acid, or4-(tert-butoxy)-2-methyl-4-oxobutanoic acid). Step 1 can take place inan organic solvent (e.g., dichloromethane) in the presence of, e.g.,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,N,N-diisopropylethylamine and DMAP. Next, halogenated diester b3 reactswith an amine c3 (x⁴ is an integer between 5 and 13, x⁵ is an integerbetween 1 and 5, e.g., heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate) to afford the product d3. Step 2 cantake place in an organic solvent (e.g., a mixture of CPME and MeCN), inthe presence of a base (such as an inorganic base (e.g., K₂CO₃) and acatalyst (e.g., an iodide such as KI) and an ether solvent (e.g.,cyclopentyl methyl ether), at an elevated temperature (e.g., about 90°C.).

A person of ordinary skill in the art will recognize that in the aboveschemes the order of certain steps may be interchangeable.

In certain aspects, the disclosure also includes methods of synthesizinga compound of any of Formulae (1-1), (2-1), (I-a), (A), (B), (A-1),(A-2), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2),(A-a3), (A-b), (A-b1), (A-b2), (A-b3), (A-c), and (B-c) andintermediate(s) for synthesizing the compound.

In some embodiments, the method of synthesizing a compound of thedisclosure includes reacting a compound of Formula (X2):

with R¹—Br to afford the compound of the disclosure, wherein eachvariables are as defined herein. For example, m is 5, 6, 7, 8, or 9,preferably 5, 7, or 9. For example, each of R⁵, R⁶, and R⁷ is H. Forexample, M is —C(O)O— or —OC(O)—. For example, R⁴ is unsubstituted C₁₋₃alkyl, or —(CH₂)_(n)Q, in which n is 2, 3, or 4 and Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, or —N(R)S(O)₂R. For example, thereaction of the compound of Formula (X2) with R¹—Br takes place in thepresence of a base (such as an inorganic base (e.g., K₂CO₃) ornon-nucleophilic organic base (e.g., i-Pr2EtN)). For example, thereaction takes place in the presence of an inorganic base (e.g., K₂CO₃)and a catalyst (e.g., an iodide such as KI or NaI). For example, thereaction takes place at an elevated temperature, e.g., about 50-100° C.,70-90° C., or about 80° C.).

The method may also include reacting a compound of Formula (X1):

with R⁴NH₂ to afford a compound of Formula (X2), wherein each variablesare as defined herein.

In some embodiments, the intermediate(s) include those having any ofFormulae (X1) and (X2):

wherein each variables are as defined herein. For example, theintermediate includes heptadecan-9-yl 8-bromooctanoate, andheptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate, and morphic formsthereof (e.g., a crystalline form).

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES Example 1: Synthesis of Compounds of Table 1 A. GeneralConsiderations

All solvents and reagents used were obtained commercially and used assuch unless noted otherwise. ¹H NMR spectra were recorded in CDCl₃, at300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts arereported as parts per million (ppm) relative to TMS (0.00) for ¹H.Silica gel chromatographies were performed on ISCO CombiFlash Rf+ LumenInstruments using ISCO RediSep Rf Gold Flash Cartridges (particle size:20-40 microns). Reverse phase chromatographies were performed on ISCOCombiFlash Rf+ Lumen Instruments using RediSep Rf Gold C18 HighPerformance columns. All final compounds were determined to be greaterthan 85% pure via analysis by reverse phase UPLC-MS (retention times,RT, in minutes) using Waters Acquity UPLC instrument with DAD and ELSDand a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column,2.1 mm, 50 mm, 1.8 μm, and a gradient of 65 to 100% acetonitrile inwater with 0.1% TFA over 5 minutes at 1.2 mL/min. Injection volume was 5μL and the column temperature was 80° C. Detection was based onelectrospray ionization (ESI) in positive mode using Waters SQD massspectrometer (Milford, Mass., USA) and evaporative light scatteringdetector.

The procedures described below are useful in the synthesis of compoundsof Table 1.

The following abbreviations are employed herein:

THF: Tetrahydrofuran

MeCN: Acetonitrile

LAH: Lithium Aluminum Hydride

DCM: Dichloromethane

DMAP: 4-Dimethylaminopyridine

LDA: Lithium Diisopropylamide

rt: Room Temperature

DME: 1,2-Dimethoxyethane

n-BuLi: n-Butyllithium

CPME: Cyclopentyl methyl ether

i-Pr₂EtN: N,N-Diisopropylethylamine

AA. Compound 6: Heptadecan-9-yl8-42-hydroxyethyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate

UPLC/ELSD: RT=3.12 min. MS (ES): m/z (MH⁺) 713.191 for C₄₃H₈₅NO₆ ¹HNMR(300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.17 (t, 4H); 3.56 (bm, 2H);2.75-2.36 (m, 6H); 2.30 (t, 2H); 1.77-1.20 (m, 61H); 0.90 (m, 9H).

AB. Compound 25: Heptadecan-9-yl8-43-42-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate6-Bromohexyl (4-nitrophenyl) carbonate

4-Nitrophenyl chloroformate (9.35 g, 46.39 mmol) was added in oneportion to a solution of 6-bromo-1-hexanol (7 g, 38.658 mmol) in DCM(100 mL) in a round bottom flask charged with a magnetic stir bar at rt.The reaction was kept under N2 and pyridine (4.69 mL, 57.988 mmol) wasadded dropwise over 10 min. after which stirring was continued at rt for16h. The reaction was diluted with water and DCM. The organic layer wasseparated, and the aqueous layer was washed with DCM. The combinedorganics were washed with brine, dried with Na₂SO₄ and evaporated undervacuum. The residue was purified by silica gel chromatography (0-100%ethyl acetate in hexanes) to give 6-bromohexyl (4-nitrophenyl) carbonate(10.3 g, 38.7 mmol, 77%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 8.29 (d, 2H);7.43 (d, 2H); 4.33 (t, 2H); 3.43 (t, 2H); 2.02-1.69 (m, 4H); 1.53 (m,4H).

6-Bromohexyl nonyl carbonate

Nonan-1-ol (8.334 g, 57.774 mmol) was added dropwise to a solution of6-bromohexyl 4-nitrophenyl carbonate (5 g, 14.443 mmol) in DCM (15 mL)in a round bottom flask charged with a magnetic stir bar at rt under N2.The reaction was kept under N₂ and pyridine (1.46 mL, 18.054 mmol) wasadded dropwise over 10 min. followed by 4-dimethylaminopyridine (0.353g, 2.889 mmol) in one portion. The reaction was allowed to stir at rtfor 16 h then diluted with water and DCM. The organic layer wasseparated, and the aqueous layer was washed with DCM. The combinedorganics were washed with brine, dried with Na₂SO₄. and evaporated undervac. The residue was purified by silica gel chromatography (0-100% ethylacetate in hexanes) to give 6-bromohexyl nonyl carbonate (1.3 g, 3.7mmol, 26%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.15 (m, 4H); 3.43 (t, 2H);1.89 (m, 2H); 1.71 (m, 4H); 1.58-1.18 (m, 16H); 0.90 (m, 3H).

Heptadecan-9-yl8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate

A solution of heptadecan-9-yl 8-bromooctanoate (69.2 g, 0.15 mole) andtert-butyl (3-aminopropyl)carbamate (130.6 g, 0.75 mole) in 500 mLethanol was heated to 65° C. overnight. The reaction mixture wasconcentrated, and the crude was purified by flash column chromatography(SiO₂: methanol/dichloromethane 0-20%) to get heptadecan-9-yl8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate (62 g, 74%) aslight yellow oil. MS (CI): m/z (MH⁺) 555.5 for C₃₃H₆₆N₂O₄. ¹H NMR (300MHz, CDCl₃): δ ppm 5.15 (bs, 1H); 4.85 (quint., 1H, J=6.0 Hz); 3.17 (m,2H); 2.65 (t, 2H, J=6.6 Hz); 2.56 (t, 2H, J=6.8 Hz); 2.26 (t, 2H, J=7.6Hz); 1.68-1.56 (m, 6H); 1.46 (m, 5H); 1.43 (s, 9H); 1.24 (m, 30H); 0.86(t, 6H, J=6.6 Hz).

Heptadecan-9-yl8-((3-((tert-butoxycarbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate

To a solution of 6-bromohexyl nonyl carbonate (0.633 g, 1.802 mmol) andheptadecan-9-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate(1 g, 1.802 mmol) in 40 mL of a 1:1 mixture of cyclopentyl methyl etherand acetonitrile was added potassium carbonate (0.996 g, 7.208 mmol) andpotassium iodide (0.329 g, 1.982 mmol). The reaction was heated to 77°C. and stirred for 16 h. The reaction was cooled, filtered and thevolatiles were evaporated under vacuum. The residue was purified bysilica gel chromatography (0-100% (a solution of 20% MeOH, 80% DCM, 1%NH₄OH) in DCM) to give heptadecan-9-yl8-((3-((-butoxycarbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate(0.32 g, 0.39 mmol, 22%). UPLC/ELSD: RT=3.02 min. MS (ES): m/z (MH⁺)826.300 for C₄₉H₉₆N₂O₇. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.65 (bm, 1H);4.88 (m, 1H); 4.14 (t, 4H); 3.19 (bm, 2H); 3.00 (bm, 1H); 2.52-2.22 (m,7H); 1.76-1.17 (m, 71H); 0.90 (m, 9H).

Heptadecan-9-yl8-((3-aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate

To a solution of heptadecan-9-yl8-43-((tert-butoxycarbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate(320 mg, 0.39 mmol) in 3 mL DCM was added trifluoroacetic acid (0.6 mL,7.8 mmol) and the solution stirred at room temp for 16 hours. Thereaction was quenched by adding a saturated sodium bicarbonate solutionat 0° C. The organic layer was washed with a saturated sodiumbicarbonate solution and brine. After drying with anhydrous sodiumsulfate, the solvent was removed under vacuum to give heptadecan-9-yl8-43-aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyDamino)octanoate (283mg, quant). UPLC/ELSD: RT=2.39 min. MS (ES): m/z (MH⁺) 726.142 forC₄₄H₈₈N₂O₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.87 (m, 1H); 4.14 (m, 4H);3.21-2.97 (m, 4H); 2.78 (m, 4H); 2.30 (m, 2H); 2.04 (m, 2H); 1.80-1.17(m, 60H); 0.90 (m,9H).

3-Methoxy-4-(methylamino)cyclobut-ene-1,2-dione

To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in100 mL diethyl ether was added a 2M methylamine solution in THF (3.8 mL,7.6 mmol) and a ppt. formed almost immediately. The mixture was stirredat rt for 24 hours, then filtered, the filter solids washed with diethylether and air-dried. The filter solids were dissolved in hot EtOAc,filtered, the filtrate allowed to cool to room temp., then cooled to 0°C. to give a ppt. This was isolated via filtration, washed with coldEtOAc, air-dried, then dried under vacuum to give3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.70 g, 5 mmol, 73%)as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ: ppm 8.50 (br. d, 1H, J=69Hz); 4.27 (s, 3H); 3.02 (sdd, 3H, J=42 Hz, 4.5 Hz).

Heptadecan-9-yl8-43-42-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate (0.2g, 0.28 mmol) in THF (2 mL) and water (0.4 mL) was added3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.06 g, 0.41 mmol),the reaction heated to 67° C. and stirred for 20 h. The reaction wasallowed to cool to rt during which the clear solution became a turbidmixture. Anhydrous MgSO₄ was added, the reaction was filtered and thefiltrate conc. The residue was purified by silica gel chromatography(0-100% (a solution of 20% MeOH, 80% DCM, 1% NH₄OH) in DCM) to giveheptadecan-9-yl8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate(120 mg, 0.14 mmol, 50%). UPLC/ELSD: RT=2.81 min. MS (ES): m/z (MH⁺)835.058 for C₄₉H₉₁N₃O₇. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.88 (p, 1H);4.14 (m, 4H); 3.74 (m, 2H); 3.40-2.90 (m, 9H); 2.31 (m, 2H); 2.12 (m,2H); 1.80-1.20 (m, 62H); 0.90 (m, 9H).

AC. Compound 64: Heptadecan-9-yl8-((2-hydroxyethyl)(6-((((3-pentyloctyl)oxy)carbonyl)oxy)hexyl)amino)octanoate

UPLC/ELSD: RT=3.04 min. MS (ES): m/z (MH⁺) 769.313 for C₄₇H₉₃NO₆ ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.14 (m, 4H); 3.54 (bm, 2H);2.66-2.37 (m, 6H); 2.30 (m, 2H); 1.77-1.17 (m, 66H); 0.91 (m, 12H).

AD. Compound 65: Heptadecan-9-yl8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)amino)octanoateEthyl 3-hexylnon-2-enoate

Triethyl phosphonoacetate (26.33 g, 117.4 mmol) was added dropwise over20 minutes to a suspension of sodium hydride (4.697 g, 117.4 mmol) inTHF (294 mL) and the mixture was stirred at room temperature until gasevolution ceased (approximately 30 min). The reaction mixture waschilled to 0° C. and 7-tridecanone (10 g, 58.7 mmol) was added. Thereaction was gradually warmed to room temperature, then heated to refluxand stirred overnight. The reaction was quenched with saturated aqueoussodium bicarbonate. The aqueous phase was extracted with diethyl ether,and the organic extracts were washed with brine, dried with MgSO₄, andconcentrated. The crude material was purified by silica gelchromatography (0-20% EtOAc:hexanes) to afford ethyl 3-hexylnon-2-enoate(6.7 g, 27.9 mmol, 47.5%) as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.63 (s, 1H); 4.15 (q, 2H); 2.61 (t, 2H); 2.15 (t, 2H); 1.53-1.20 (m,19H); 0.91 (m, 6H).

Ethyl 3-hexylnonanoate

To a flask containing a slurry of Pearlmans catalyst (0.73 g, 5.2 mmol)in ethanol (20 mL) under N₂ was added a solution of ethyl3-hexylnon-2-enoate (6.975 g, 25.9 mmol) in ethanol (5 mL). The reactionwas stirred under H₂ (balloon) for 16 h. The reaction was filteredthrough a plug of Celite and the filtrate was evaporated under vacuum toafford ethyl 3-hexylnonanoate (6.7 g, 24.7 mmol, 95%). The residue wastaken to the next step without further purification. ¹H NMR (300 MHz,CDCl₃) δ: ppm 4.16 (q, 2H); 2.23 (d, 2H); 1.86 (bs, 1H); 1.28 (m, 23H);0.90 (m, 6H).

3-Hexylnonan-1-ol

To a solution of lithium aluminium hydride (49.5 mL of 1M solution inTHF, 49.5 mmol) in THF was added a solution of ethyl 3-hexylnonanoate(6.7 g, 24.7 mmol) in THF (20 mL). The reaction was stirred at rt for 16h. The reaction was quenched with a saturated solution of sodium sulfatedecahydrate. The white solids were removed by filtration through a plugof Celite and the filtrate was evaporated under vacuum. The residue waspurified by flash chromatography (ISCO) by 0-100% ethyl acetate inhexanes to obtain 3-hexylnonan-1-ol (5.62 g, 24.6 mmol, 99%). ¹H NMR(300 MHz, CDCl₃) δ: ppm 3.69 (t, 2H); 1.61-1.19 (m, 24H); 0.91 (m, 6H).

Heptadecan-9-yl8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)amino)octanoate

Compound 65 was prepared analogously to compound 25 using3-hexylnonan-1-ol instead of nonan-1-ol. UPLC/ELSD: RT=3.13 min. MS(ES): m/z (MH⁺) 919.429 for C₅₅H₁₀₃N₃O₇. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.87 (p, 1H); 4.17 (m, 4H); 3.68 (bm, 2H); 3.30 (m, 3H); 2.65-2.41 (m,6H); 2.31 (m, 2H); 1.87-1.19 (m, 73H); 0.90 (m, 12H).

AE. Compound 66: Heptadecan-9-yl8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(2-hydroxyethyl)amino)octanoate

UPLC/ELSD: RT=3.16 min. MS (ES): m/z (MH⁺) 797.683 for C₄₉H₉₇NO₆ ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.14 (m, 4H); 3.54 (bm, 2H);2.68-2.38 (m, 6H); 2.30 (m, 2H); 1.77-1.17 (m, 70H), 0.90 (m, 12H).

AF. Compound 67: Heptadecan-9-yl8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-((((3-pentyloctyl)oxy)carbonyl)oxy)hexyl)amino)octanoate

Compound 67 was prepared analogously to compound 65 using 6-undecanoneinstead of 7-tridecanone. UPLC/ELSD: RT=3.04 min. MS (ES): m/z (MH⁺)891.552 for C₅₃H₉₉N₃O₇. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.87 (p, 1H);4.17 (m, 4H); 3.67 (bm, 2H); 3.28 (m, 3H); 2.68-2.38 (m, 6H); 2.31 (m,2H); 1.86-1.18 (m, 69H); 0.90 (m, 12H).

Example 2: Expression of hEPO Induced by Sample Formulations in Mice andResidual Lipid Levels in the Liver

To assess potency of expression and metabolic stability of lipids of thedisclosure the hepatocyte protein expression (hEPO) followingadministration of a nanoparticle of the disclosure (e.g., a loaded LNP)to mice was measured.

Lipid nanoparticles (LNPs) including DSPC as a phospholipid, cholesterolas a structural lipid, PEG-1 as a PEG lipid, a compound according toFormula (A), (A1), (A2), (A3), (B), (I), (I′), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa), or (IIb),and an mRNA encoding hEPO were intravenously administered to CD-1 mice.The concentration of hEPO in serum was tested at 6 h after injection.The particles tested had a PDI of between about 0.1-0.4, anencapsulation efficiency of between about 82-99%, and a particlediameter of about 56-145 nm. All of the tested LNPs demonstratedeffective delivery of mRNA to hepatocytes with varying amounts of lipidremaining in the livers of the mice after 24 h.

TABLE 2 Epression of hEPO induced by administration of LNPs comprisinglipids of the disclosure in mice and residual lipid levels in the liver.Mean 6 hr Parent Lipid Approx. Percent Ionizable hEPO Remaining TotalDose of Lipid Concen- (nmol/g Parent Lipid Compound tration hEPO livertissue) Remaining No. (mIU/mL) AUC at 24 h at 24 h 6 509181.4 8.19E+06 3± 1    2% 64 735556.2 1.11E+07 72 ± 17   51% 66 641899.1 9.77E+06 67 ±8    48% 67 891982.2 1.31E+07 94 ± 15 >95% 65 521341.6 7.76E+06 364 ±58  >95% ^(a)The percent of total dose calculation assumes a 25 g mousewith a 1.5 g liver ^(b) >95% equals very slowly metabolism of the lipid

Enumerated Embodiments

-   Embodiment 1. A compound of Formula (I1):

or its N-oxide,

or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*′, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*″, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from the group consisting of hydrogen, a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —(CH₂)_(o)C(R¹²)₂(CH₂)_(n-o)Q, —CHQR,—CQ(R)₂, —C(O)NQR and unsubstituted C₁₋₆ alkyl, where Q is selected froma carbocycle, heterocycle,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR,

-   —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R,    —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R) R⁸, —N(R)S(O)₂R⁸,    −O(CH₂)_(n)OR, —N(R)C(═NR⁹)N(R)₂, —N(R)C(═CHR⁹)N(R)₂, —OC(O)N(R)₂,    —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,    —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR⁹)N(R)₂,    —N(OR)C(═CHR⁹)N(R)₂, —C(═NR⁹)N(R)₂, —C(═NR⁹)R, —C(O)N(R)OR,-   —(CH₂)_(n)N(R)₂, —C(R)N(R)₂C(O)OR, NC(R)═R¹¹, N(C═NR¹⁵)R¹¹,    NRC(C(O)N R¹⁴R¹⁴′)₂, —NRC(O)(CH₂)_(p)C(O)N R¹⁴R¹⁴′, and

wherein A is C₆₋₁₀ aryl or a heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3,or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1,2, 3, 4, and 5; or

R⁴ is

wherein

X^(a) and X^(b) are each independently O or S;

R¹⁰ is selected from the group consisting of H, halo, —OH, R, —N(R)₂,—CN, —N₃, —C(O)OH, —C(O)OR, —OC(O)R, —OR, —SR, —S(O)R, —S(O)OR,—S(O)₂OR, —NO₂, —S(O)₂N(R)₂, —N(R)S(O)₂R, —NH(CH₂)_(t1)N(R)₂,—NH(CH₂)_(p1)O(CH₂)_(q1)N(R)₂, —NH(CH₂)_(s1)OR, —N((CH₂)_(s1)OR)₂,—N(R)-carbocycle, —N(R)-heterocycle, —N(R)-aryl, —N(R)-heteroaryl,—N(R)(CH₂)_(t1)-carbocycle, —N(R)(CH₂)_(t1)-heterocycle,—N(R)(CH₂)_(t1)-aryl, —N(R)(CH₂)_(t1)-heteroaryl, a carbocycle, aheterocycle, aryl and heteroaryl;

-   -   n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   r is 0 or 1;    -   t¹ is selected from 1, 2, 3, 4, and 5;    -   p¹ is selected from 1, 2, 3, 4, and 5;    -   q¹ is selected from 1, 2, 3, 4, and 5; and    -   s¹ is selected from 1, 2, 3, 4, and 5;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M is selected from —OC(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,SO—, —OS—, —S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, in which M″ isa bond,—(CH₂)_(z)C(O)—, C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—,—S(R**)₂—, or —S(O)—, wherein z is 1, 2, 3, or 4;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(RM)C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R⁸ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R⁹ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl,—OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

R¹¹ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle, wherein the C₃₋₆ carbocycle and heterocycle are eachoptionally substituted with one or more R¹³;

R¹² is selected from the group consisting of H, OH, C₁₋₃ alkyl, and C₂₋₃alkenyl;

each R¹³ is selected from the group consisting of OH, oxo, halo, C₁₋₆alkyl, C₁₋₆ alkoxyl, C₂₋₆ alkenyl, C₁₋₆ alkylamino, di-(C₁₋₆ alkyl)amino, amino, amido, cyano, and nitro;

each R¹⁴ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

each R¹⁴′ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

R¹⁵ is independently selected from the group consisting of H, OH, C₁₋₆alkyl, and C₂₋₃ alkenyl;

each R is independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkyl-aryl, C₂₋₃ alkenyl, and H;

R^(N) is H, or C₁₋₃ alkyl;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

-   Embodiment 2. A compound of Formula (I′1):

or its N-oxide,

or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*′, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from the group consisting of hydrogen, a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —(CH₂)_(o)C(R¹²)₂(CH₂)_(n-o)Q, —CHQR,—CQ(R)₂, —C(O)NQR and unsubstituted C₁₋₆ alkyl, where Q is selected froma carbocycle, heterocycle,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR,

-   —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R,    —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R) R⁸, —N(R)S(O)₂R⁸,    —O(CH₂)_(n)OR, —N(R)C(═NR⁹)N(R)₂, —N(R)C(═CHR⁹)N(R)₂, —OC(O)N(R)₂,    —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,    —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR⁹)N(R)₂,    —N(OR)C(═CHR⁹)N(R)₂, —C(═NR⁹)N(R)₂, —C(═NR⁹)R, —C(O)N(R)OR,-   —(CH₂)_(n)N(R)₂, —C(R)N(R)₂C(O)OR, NC(R)═R¹¹, N(C═NR¹⁵)R¹¹,    NRC(C(O)N R¹⁴R¹⁴′)₂, —NRC(O)(CH₂)_(p)C(O)N R¹⁴R¹⁴′, and

wherein A is C₆₋₁₀ aryl or a heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3,or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1,2, 3, 4, and 5; or

R⁴ is

wherein

X^(a) and X^(b) are each independently O or S;

R¹⁰ is selected from the group consisting of H, halo, —OH, R, —N(R)₂,—CN, —N₃, —C(O)OH, —C(O)OR, —OC(O)R,—OR,—SR, —S(O)R, —S(O)OR,—S(O)₂OR,—NO₂, —S(O)₂N(R)₂, —N(R)S(O)₂R, —NH(CH₂)_(ti)N(R)₂,—NH(CH₂)_(p1)O(CH₂)_(q1)N(R)₂, —NH(CH₂)_(s1)OR, —N((CH₂)_(s1)OR)₂,—N(R)-carbocycle, —N(R)-heterocycle, —N(R)-aryl, —N(R)-heteroaryl,—N(R)(CH₂)_(t1)-carbocycle, —N(R)(CH₂)_(t1)-heterocycle,—N(R)(CH₂)_(t1)-aryl, —N(R)(CH₂)_(t1)-heteroaryl, a carbocycle, aheterocycle, aryl and heteroaryl;

-   -   n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   r is 0 or 1;    -   t¹ is selected from 1, 2, 3, 4, and 5;    -   p¹ is selected from 1, 2, 3, 4, and 5;    -   q¹ is selected from 1, 2, 3, 4, and 5; and    -   s¹ is selected from 1, 2, 3, 4, and 5;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—CO)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

M′ is selected from —OC(O)O—,—OC(O)—NR^(M)-C(O)O—, —O-M″—O—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,SO—, —OS—, —S(R^(M))₂O—, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, in which M″ isa bond,—(CH₂)_(z)C(O)—, C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—,—S(R**)₂—, or —S(O)—, wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R⁸ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R⁹ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

R¹¹ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle, wherein the C₃₋₆ carbocycle and heterocycle are eachoptionally substituted with one or more R¹³;

R¹² is selected from the group consisting of H, OH, C₁₋₃ alkyl, and C₂₋₃alkenyl;

each R¹³ is selected from the group consisting of OH, oxo, halo, C₁₋₆alkyl, C₁₋₆ alkoxyl, C₂₋₆ alkenyl, C₁₋₆ alkylamino, di-(C₁₋₆ alkyl)amino, amino, amido, cyano, and nitro;

each R¹⁴ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

each R¹⁴′ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

R¹⁵ is independently selected from the group consisting of H, OH, C₁₋₆alkyl, and C₂₋₃ alkenyl;

each R is independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkyl-aryl, C₂₋₃ alkenyl, and H;

R^(N) is H, or C₁₋₃ alkyl;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

-   Embodiment 3. A compound of Formula (IX1):

or its N-oxide, or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*″, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*′, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from the group consisting of hydrogen, a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —(CH₂)_(o)C(R¹²)₂(CH₂)_(n-o)Q, —CHQR,—CQ(R)₂, —C(O)NQR and unsubstituted C₁₋₆ alkyl, where Q is selected froma carbocycle, heterocycle,—OR, −O(CH₂)_(n)N(R)₂, —C(O)OR,

-   —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R,    —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R) R⁸, —N(R)S(O)₂R⁸,    —O(CH₂)_(n)OR, —N(R)C(═NR⁹)N(R)₂, —N(R)C(═CHR⁹)N(R)₂, —OC(O)N(R)₂,    —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,    —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR⁹)N(R)₂,    —N(OR)C(═CHR⁹)N(R)₂, —C(═NR⁹)N(R)₂, —C(═NR⁹)R, —C(O)N(R)OR,-   —(CH₂)_(n)N(R)₂, —C(R)N(R)₂C(O)OR, NC(R)═R¹¹, N(C═NR15,)R¹¹,    NRC(C(O)N R¹⁴R¹⁴)₂, —NRC(O)(CH₂)_(p)C(O)N R¹⁴R¹⁴′, and

wherein A is C₆₋₁₀ aryl or a heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3,or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1,2, 3, 4, and 5; or

R⁴ is

wherein

X^(a) and X^(b) are each independently O or S;

R¹⁰ is selected from the group consisting of H, halo, —OH, R,—N(R)₂,—CN, —N₃, —C(O)OH, —C(O)OR, —OC(O)R, —OR, —SR, —S(O)R, —S(O)OR,—S(O)₂OR, —NO₂, —S(O)₂N(R)₂, —N(R)S(O)₂R, —NH(CH₂)_(t1)N(R)₂,—NH(CH₂)_(p1)O(CH₂)_(q1)N(R)₂, —NH(CH₂)_(s1)OR, —N((CH₂)_(s1)OR)₂,—N(R)-carbocycle, —N(R)-heterocycle, —N(R)-aryl, —N(R)-heteroaryl,—N(R)(CH₂)_(t1)-carbocycle, —N(R)(CH₂)_(t1)-heterocycle,—N(R)(CH₂)_(t1)-aryl, —N(R)(CH₂)_(t1)-heteroaryl, a carbocycle, aheterocycle, aryl and heteroaryl;

-   -   n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   r is 0 or 1;    -   t¹ is selected from 1, 2, 3, 4, and 5;    -   p¹ is selected from 1, 2, 3, 4, and 5;    -   q¹ is selected from 1, 2, 3, 4, and 5; and    -   s¹ is selected from 1, 2, 3, 4, and 5;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,—OC(O)—NR^(M)—C(O)O—, —O-M″—O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R⁸ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R⁹ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

R¹¹ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle, wherein the C₃₋₆ carbocycle and heterocycle are eachoptionally substituted with one or more R¹³;

R¹² is selected from the group consisting of H, OH, C₁₋₃ alkyl, and C₂₋₃alkenyl;

each R¹³ is selected from the group consisting of OH, oxo, halo, C₁₋₆alkyl, C₁₋₆ alkoxyl, C₂₋₆ alkenyl, C₁₋₆ alkylamino, di-(C₁₋₆ alkyl)amino, amino, amido, cyano, and nitro;

each R¹⁴ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

each R¹⁴′ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

R¹⁵ is independently selected from the group consisting of H, OH, C₁₋₆alkyl, and C₂₋₃ alkenyl;

each R is independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkyl-aryl, C₂₋₃ alkenyl, and H;

R^(N) is H, or C₁₋₃ alkyl;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

-   Embodiment 4. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —OC(O)O—.-   Embodiment 5. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —OC(O)N(R^(M))—, or    —N(R^(M))C(O)O—.-   Embodiment 6. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —NR^(M)C(O)NR^(M)—.-   Embodiment 7. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —O—N═C(R^(M))—.-   Embodiment 8. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —O-M″—O—, in which each M″ is    independently selected from C₁₋₁₃ alkyl, C₂₋₁₃ alkenyl, —B(R**)—,    and —Si(R**)₂—.-   Embodiment 9. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —O-M″—O—, and M″ is a C₁₋₃    alkyl.-   Embodiment 10. The compound any one of the preceding embodiments,    wherein M″ is methyl.-   Embodiment 11. The compound any one of the preceding embodiments,    wherein M″ is a branched C₁₋₃ alkyl.-   Embodiment 12. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —O-M″—O—, and M″ is    —(CH₂)_(z)C(O)—.-   Embodiment 13. The compound of any one of the preceding embodiments,    wherein z is 3.-   Embodiment 14. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is

-   Embodiment 15. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is

-   Embodiment 16. The compound of any one of the preceding embodiments,    wherein at least one of M and M′ is —O-M″—O—, in which each M″ is    independently selected from —B(R**)— and —Si(R**)₂—.-   Embodiment 17. The compound of any one of the preceding embodiments,    wherein each R** is a C₁₋₃ alkyl.-   Embodiment 18. The compound of any one of the preceding embodiments,    wherein each R** is methyl.-   Embodiment 19. The compound of any one of the preceding embodiments,    wherein one of M and M′ is —NR^(M)C(O)NR^(M)—.-   Embodiment 20. The compound of any one of the preceding embodiments,    wherein each R^(M) is H.-   Embodiment 21. The compound of any one of the preceding embodiments    wherein R¹ is R″M′R′ and R′ is a branched alkyl.-   Embodiment 22. The compound of any one of the preceding embodiments,    wherein R¹ is R″M′R′ and R′ is an alkenyl.-   Embodiment 23. The compound of any one of the preceding embodiments,    wherein R¹ is R″M′R′ and R″ is a branched alkyl.-   Embodiment 24. The compound of any one of the preceding embodiments,    wherein R¹ is R″M′R′ and R″ is an alkyl substituted with OH.-   Embodiment 25. The compound of any one of the preceding embodiments,    wherein R′ is R*YR*″ and Y is a cyclopropyl, cyclopentyl, or    cyclohexyl.-   Embodiment 26. The compound of any one of the preceding embodiments,    wherein R′ is R*YR*″ and R*″ is a C₂ alkyl, a C3-alkyl, or a C₄    alkyl.-   Embodiment 27. The compound of any one of the preceding embodiments,    wherein the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 28. The compound of any one of the preceding embodiments,    wherein the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 29. The compound of one of the preceding claims, wherein    the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 30. The compound of one of the preceding claims, wherein    the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 31. The compound of one of the preceding claims, wherein    the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 32. The compound of any one of the preceding embodiments    1 or 2, wherein the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 33. The compound of any one of the preceding embodiments,    wherein the compound has the following structure:

wherein R″ is a linear or branched C₃-C₁₃ alkyl, optionally substitutedwith OH, and m is selected from 5, 6, 7, 8, and 9.

-   Embodiment 34. A compound of Formula (I-Y1):

or its N-oxide, or a salt or isomer thereof, wherein:

R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR*′, —YR*″, and —R″M′R′;

R² and R³ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR*′, —YR*″, and —R*OR*″, or R² and R³,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R⁴ is selected from the group consisting of hydrogen, a C₃₋₆ carbocycle,—(CH₂ _(n)Q, —(CH₂)_(n)CHQR, —(CH₂)_(o)C(R¹²)₂(CH₂)_(n-o)Q, —CHQR,—CQ(R)₂, —C(O)NQR and unsubstituted C₁₋₆ alkyl, where Q is selected froma carbocycle, heterocycle,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR,

-   —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R,    —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R) R⁸, —N(R)S(O)₂R⁸,    —O(CH₂)_(n)OR, —N(R)C(═NR⁹)N(R)₂, —N(R)C(═CHR⁹)N(R)₂, —OC(O)N(R)₂,    —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,    —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR⁹)N(R)₂,    —N(OR)C(═CHR⁹)N(R)₂, —C(═NR⁹)N(R)₂, —C(═NR⁹)R, —C(O)N(R)OR,-   —(CH₂)_(n)N(R)₂, —C(R)N(R)₂C(O)OR, NC(R)═R¹¹, N(C═NR¹⁵)R¹¹,    NRC(C(O)N R¹⁴R¹⁴′)₂, —NRC(O)(CH₂)_(p)C(O)N R¹⁴R¹⁴′, and

wherein A is C₆₋₁₀ aryl or a heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3,or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1,2, 3, 4, and 5; or

R⁴ is

wherein

X^(a) and X^(b) are each independently O or S;

R¹⁰ is selected from the group consisting of H, halo, —OH, R,—N(R)₂,—CN, —N₃, —C(O)OH, —C(O)OR, —OC(O)R,—OR,—SR, —S(O)R, —S(O)OR,—S(O)₂OR, —NO₂, —S(O)₂N(R)₂, —N(R)S(O)₂R, —NH(CH₂)_(t1)N(R)₂,—NH(CH₂)_(p1)O(CH₂)_(q1)N(R)₂, —NH(CH₂)_(s1)OR, —N((CH₂)_(s1)OR)₂,—N(R)-carbocycle, —N(R)-heterocycle, —N(R)-aryl, —N(R)-heteroaryl,—N(R)(CH₂)_(t1)-carbocycle, —N(R)(CH₂)_(t1)-heterocycle,—N(R)(CH₂)_(t1)-aryl, —N(R)(CH₂)_(t1)-heteroaryl, a carbocycle, aheterocycle, aryl and heteroaryl;

-   -   n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   r is 0 or 1;    -   t¹ is selected from 1, 2, 3, 4, and 5;    -   p¹ is selected from 1, 2, 3, 4, and 5;    -   q¹ is selected from 1, 2, 3, 4, and 5; and    -   s¹ is selected from 1, 2, 3, 4, and 5;

each R⁵ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R⁶ is independently selected from the group consisting of OH, C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M′ is selected from —OC(O)O—, —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—,—OC(O)—NR^(M)-C(O)O—, —O-M″-O—, —C(O)N(R^(M))—, —N(R^(M))C(O)—,—OC(O)N(R^(M))—, —N(R^(M))C(O)O—, —NR^(M)C(O)NR^(M)—, —O—N═C(R^(M))—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR^(M))O—, —S(O)₂—,—S—S—, —SO—, —OS—, S(R^(M))₂O, —O—S(R^(M))₂—, —S(O)O—, —OS(O)—, an arylgroup, and a heteroaryl group, in which M″ is a bond,—(CH₂)_(z)C(O)—,C₁₋₁₃ alkyl,C₂₋₁₃ alkenyl, —B(R**)—, —Si(R**)₂—, —S(R**)₂—, or —S(O)—,wherein z is 1, 2, 3, or 4;

R⁷ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R⁸ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R⁹ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl,—OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

R¹¹ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle, wherein the C₃₋₆ carbocycle and heterocycle are eachoptionally substituted with one or more R¹³;

R¹² is selected from the group consisting of H, OH, C₁₋₃ alkyl, and C₂₋₃alkenyl;

each R¹³ is selected from the group consisting of OH, oxo, halo, C₁₋₆alkyl, C₁₋₆ alkoxyl, C₂₋₆ alkenyl, C₁₋₆ alkylamino, di-(C₁₋₆ alkyl)amino, amino, amido, cyano, and nitro;

each R¹⁴ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

each R¹⁴′ is independently selected from the group consisting of H, OH,C₁₋₆ alkyl, and C₂₋₃ alkenyl;

R¹⁵ is independently selected from the group consisting of H, OH, C₁₋₆alkyl, and C₂₋₃ alkenyl;

each R is independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkyl-aryl, C₂₋₃ alkenyl, and H;

R^(N) is H, or C₁₋₃ alkyl;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR*″, —YR*″, (CH₂)_(q)OR*, and H,

and each q is independently selected from 1, 2, and 3;

each R^(M) is independently selected from the group consisting of H,C₁₋₆ alkyl and C₂₋₆ alkenyl;

each R″ is independently selected from the group consisting of C₃₋₁₅alkyl and C₃₋₁₅ alkenyl;

each R*″ is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R** is independently selected from the group consisting of H, OH,C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, (CH₂)_(q)OR*, and (CH₂)_(q)OH;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

-   Embodiment 35. The compound of any one of the preceding embodiments,    wherein the compound has the following structure:

wherein each R″ independently is selected from H, OH, and C₁₋₆ alkyl, 1is selected from 2, 3, 4, 5, and 6, and m is selected from 5, 6, 7, 8,and 9.

-   Embodiment 36. The compound of any one of the preceding embodiments,    wherein R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from    1, 2, 3, 4, and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

-   Embodiment 37. A compound of Formula (A):

or its N-oxide, or a salt or isomer thereof, wherein

m is selected from 5, 6, 7, 8, and 9;

R² and R³ are each independently selected from the group consisting ofH, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl;

R⁴ is selected from —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4,and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H;

M and M′ are each independently selected from —OC(O)O— and —C(O)O—;

wherein at least one of M and M′ is —OC(O)O—;

R′^(a) is C₁₋₁₈ alkyl or C₂₋₁₈ alkenyl; and

R″ is a C₃-C₁₃ alkyl, optionally substituted with OH.

-   Embodiment 38. The compound of any one of the preceding embodiments,    wherein R″ is selected from

wherein

denotes a point of attachment with N, and

denotes a point of attachment with M.

-   Embodiment 39. The compound of any one of the preceding embodiments,    wherein the compound has one of the following structures:

-   Embodiment 40. The compound of any one of the preceding embodiments,    wherein R′^(a) is:

wherein

denotes a point of attachment;

R^(aα), R^(aβ), and R^(aγ) are each independently selected from thegroup consisting of H, C₁₋₁₂ alkyl, and C₂₋₁₂ alkenyl, wherein at leastone of R^(aα), R^(aβ), and R^(aγ) is selected from the group consistingof C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; and

R′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl.

-   Embodiment 41. The compound of any one of the preceding embodiments,    wherein R^(aβ) and R^(aγ) are each H and R^(aα) is C₁₋₆ alkyl.-   Embodiment 42. The compound of any one of the preceding embodiments,    wherein R^(aα) and R^(aγ) are each H and R^(aβ) is C₁₋₆ alkyl.-   Embodiment 43. The compound of any one of the preceding embodiments,    wherein R^(aβ) is C₂ alkyl.-   Embodiment 44. The compound of any one of the preceding embodiments,    wherein R^(aα) and R^(aβ) are each H and R^(a)Y is C₁₋₆ alkyl.-   Embodiment 45. The compound of any one of the preceding embodiments,    wherein R^(aγ) is a C₅ or a C₆ alkyl.-   Embodiment 46. The compound of any one of the preceding embodiments    wherein R′ is a C₅ or a C₆ alkyl.-   Embodiment 47. The compound of any one of the preceding embodiments,    wherein the compound has the following structure:

-   Embodiment 48. The compound of any one of the preceding embodiments,    wherein R⁴ is —(CH₂)_(n)OH.-   Embodiment 49. The compound of any one of the preceding embodiments,    wherein R⁴ is —(CH₂)₂OH.-   Embodiment 50. The compound of any one of the preceding embodiments,    wherein R⁴ is

-   Embodiment 51. The compound of any one of the preceding embodiments,    wherein R⁴ is

-   Embodiment 52. The compound of any one of the preceding embodiments,    wherein n2 is 2 or 4.-   Embodiment 53. A compound selected from:

-   Embodiment 54. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid.-   Embodiment 55. A loaded lipid nanoparticle (loaded LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, a PEG lipid, and one or more therapeutic and/or    prophylactic agents.-   Embodiment 56. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising the compound in an amount from    about 40% to about 60%.-   Embodiment 57. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising the phospholipid in an amount from    about 0% to about 20%.-   Embodiment 58. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising the structural lipid in an amount    from about 30% to about 50%.-   Embodiment 59. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising the PEG lipid in an amount from    about 0% to about 5%.-   Embodiment 60. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising about 40 mol % to about 60 mol %    of the compound of any one of the preceding embodiments, about 0 mol    % to about 20 mol % phospholipid, about 30 mol % to about 50 mol %    structural lipid, and about 0 mol % to about 5 mol % PEG lipid.-   Embodiment 61. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising about 30 mol % to about 60 mol %    of the compound of any one of the preceding embodiments, about 0 mol    % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol    % structural lipid, and about 0 mol % to about 10 mol % PEG lipid.-   Embodiment 62. The loaded LNP of any one of the preceding    embodiments, wherein the one or more therapeutic and/or prophylactic    agents is a polynucleotide or a polypeptide.-   Embodiment 63. The loaded LNP of any one of the preceding    embodiments, wherein the one or more therapeutic and/or prophylactic    agents is a nucleic acid.-   Embodiment 64. The loaded LNP of any one of the preceding    embodiments, wherein the one or more therapeutic and/or prophylactic    agents is selected from the group consisting of a ribonucleic acid    (RNA) and a deoxyribonucleic acid (DNA).-   Embodiment 65. The loaded LNP of any one of the preceding    embodiments, wherein the DNA is selected from the group consisting    of a double-stranded DNA, a single-stranded DNA (ssDNA), a partially    double-stranded DNA, a triple stranded DNA, and a partially    triple-stranded DNA.-   Embodiment 66. The loaded LNP of any one of the preceding    embodiments, wherein the DNA is selected from the group consisting    of a circular DNA, a linear DNA, and mixtures thereof.-   Embodiment 67. The loaded LNP of any one of the preceding    embodiments, wherein the one or more therapeutic and/or prophylactic    agents is selected from the group consisting of a plasmid expression    vector, a viral expression vector, and mixtures thereof.-   Embodiment 68. The loaded LNP of any one of the preceding    embodiments, wherein the one or more therapeutic and/or prophylactic    agents is a RNA.-   Embodiment 69. The loaded LNP of any one of the preceding    embodiments, wherein the RNA is selected from the group consisting    of a single-stranded RNA, a double-stranded

RNA (dsRNA), a partially double-stranded RNA, and mixtures thereof.

-   Embodiment 70. The loaded LNP of any one of the preceding    embodiments, wherein the RNA is selected from the group consisting    of a circular RNA, a linear RNA, and mixtures thereof.-   Embodiment 71. The loaded LNP of any one of the preceding    embodiments, wherein the RNA is selected from the group consisting    of is selected from the group consisting of a short interfering RNA    (siRNA), an asymmetrical interfering RNA (aiRNA), a RNA interference    (RNAi) molecule, a microRNA (miRNA), an antagomir, an antisense RNA,    a ribozyme, a Dicer-substrate RNA (dsRNA), a small hairpin RNA    (shRNA), a messenger RNA (mRNA), and mixtures thereof.-   Embodiment 72. The loaded LNP of any one of the preceding    embodiments, wherein the RNA is an mRNA.-   Embodiment 73. The loaded LNP of any one of the preceding    embodiments, wherein the mRNA is a modified mRNA (mmRNA).-   Embodiment 74. The loaded LNP of any one of the preceding    embodiments, wherein the mRNA incorporates a micro-RNA binding site    (miR binding site).-   Embodiment 75. The loaded LNP of any one of the preceding    embodiments, wherein the mRNA includes one or more of a stem loop, a    chain terminating nucleoside, a polyA sequence, a polyadenylation    signal, and/or a 5′ cap structure.-   Embodiment 76. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the phospholipid is selected from the    group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine    (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),    1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),    1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),    1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),    1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),    1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),    1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),    1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine    (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),    1,2-dilinolenoyl-sn-glycero-3-phosphocholine,    1,2-diarachidonoyl-sn-glycero-3-phosphocholine,    1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoetha    nolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine    (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,    1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,    1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,    1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,    1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,    1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt    (DOPG), sphingomyelin, and mixtures thereof.-   Embodiment 77. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the phospholipid is    1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).-   Embodiment 78. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the structural lipid is selected from    the group consisting of cholesterol, fecosterol, sitosterol,    ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine,    ursolic acid, alpha-tocopherol, and mixtures thereof.-   Embodiment 79. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the structural lipid is

a salt thereof.

-   Embodiment 80. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the structural lipid is cholesterol:

or a salt thereof.

-   Embodiment 81. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is selected from the    group consisting of a PEG-modified phosphatidylethanolamine, a    PEG-modified phosphafidic acid, a PEG-modified ceramide, a.    PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a    PEG-modified dialkylglycerol, and mixtures thereof.-   Embodiment 82. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is selected from the    group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene    glycol (PEG-DMG),    1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene    glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG),    PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide    (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or    PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).-   Embodiment 83. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is PEG-DMG.-   Embodiment 84. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PL-I):

or a salt thereof, wherein:

R^(3PL1) is —OR^(OPL1);

R^(OPL1) is hydrogen, optionally substituted alkyl, or an oxygenprotecting group;

r^(PL1) is an integer between 1 and 100, inclusive;

L¹ is optionally substituted C₁₋₁₀ alkylene, wherein at least onemethylene of the optionally substituted C₁₋₁₀ alkylene is independentlyreplaced with optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted arylene, optionallysubstituted heteroarylene, O, N(R^(NPL1)), S, C(O), C(O)N(R^(NPL1)),NR^(NPL1)C(O), —C(O)O, OC(O), OC(O)O, OC(O)N(R^(NPL1)), NR^(NPL1)C(O)O,or NR^(NPL1)C(O)N(R^(NPL1));

D is a moiety obtained by click chemistry or a moiety cleavable underphysiological conditions;

m^(PL1) is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

each instance of of L² is independently a bond or optionally substitutedC₁₋₆ alkylene, wherein one methylene unit of the optionally substitutedC₁₋₆ alkylene is optionally replaced with O, N(R^(NPL1)), S, C(O),C(O)N(R^(NPL1)), RR^(NPL1)C(O), C(O)O, OC(O), OC(O)O, —OC(O)N(R^(NPL1)),NR^(NPL1)C(O)O, or NR^(NPL1)C(O)N(R^(NPL1));

each instance of R^(2SL) is independently optionally substituted C₁₋₃₀alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally substitutedC₁₋₃₀ alkynyl; optionally wherein one or more methylene units of R^(2SL)are independently replaced with optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,optionally substituted heteroarylene, N(R^(NPL1)), O, S, C(O),C(O)N(R^(NPL1)), NR^(NPL1)C(O), —NR^(NPL1)C(O)N(R^(NPL1)), C(O)O, OC(O),OC(O)O, OC(O)N(R^(NPL1)), NR^(NPL1)C(O)O, C(O)S, —SC(O), C(═NR^(NPL1)),C(═NR^(NPL1))N(R^(NPL1)), NR^(NPL1)C(═NR^(NPL1)),—NR^(NPL1)C(═NR^(NPL1))N(R^(NPL1)), C(S), C(S)N(R^(NPL1)),NR^(NPL1)C(S), NR^(NPL1)C(S)N(R^(NPL1)), S(O) OS(O), S(O)O, OS(O)O,OS(O)₂, S(O)₂O, OS(O)₂O, N(R^(NPL1))S(O), S(O)N(R^(NPL1)),—N(R^(NPL1))S(O)N(R^(NPL1)), OS(O)N(R^(NPL1)), N(R^(NPL1))S(O)O, S(O)₂,N(R^(NPL1))S(O)₂, —S(O)₂N(R^(NPL1)), N(R^(NPL1))S(O)₂N(R^(NPL1)),OS(O)₂N(R^(NPL1)), or N(R^(NPL1))S(O)₂O;

each instance of R^(NPL1) is independently hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl; and

p^(SL) is 1 or 2.

-   Embodiment 85. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PL-I-OH):

or a salt thereof.

-   Embodiment 86. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PL-II-OH):

or a salt or isomer thereof, wherein:

R^(3PEG) is-OR^(O);

R^(O) is hydrogen, C₁₋₆ alkyl or an oxygen protecting group;

r ^(PEG) is an integer between 1 and 100;

R^(5PEG) is C₁₀₋₄₀ alkyl, C₁₀₋₄₀ alkenyl, or C₁₀₋₄₀ alkynyl; andoptionally one or more methylene groups of R^(5PEG) are independentlyreplaced with C₃₋₁₀ carbocyclylene, 4 to 10 membered heterocyclylene,C₆₋₁₀ arylene, 4 to 10 membered heteroarylene,)—N(R^(NPEG))—, —O—, —S—,—C(O)—, —C(O)N(R^(NPEG))—, —NR^(NPEG)C(O)—, —NR^(NPEG)C(O)N(R^(NPEG))—,—C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(NPEG))—, —NR^(NPEG)C(O)O—,—C(O)S—, —SC(O)—, —C(═NR^(NPEG))—, —C(═NR^(NPEG))N(R^(NPEG))—,—NR^(NPEG)C(═NR^(NPEG))—, —NR^(NPEG)C(═NR^(NPEG))N(R^(NPEG))—, —C(S)—,—C(S)N(R^(NPEG))—, —NR^(NPEG)C(S)—, —NR^(NPEG)C(S)N(R^(NPEG))—, —S(O)—,—OS(O)—, —S(O)O—, —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—,—N(R^(NPEG))S(O)—, —S(O)N(R^(NPEG))—, —N(R^(NPEG))S(O)N(R^(NPEG))—,—OS(O)N(R^(NPEG))—, —N(R^(NPEG))S(O)O—, —S(O)₂—, —N(R^(NPEG))S(O)₂—,—S(O)₂N(R^(NPEG))—, —N(R^(NPEG))S(O)₂N(R^(NPEG))—, —OS(O)₂N(R^(NPEG))—,or —N(R^(NPEG))S(O)₂O—; and

each instance of R^(NPEG) is independently hydrogen, C₁₋₆ alkyl, or anitrogen protecting group.

-   Embodiment 87. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein in the PEG lipid of Formula    (PL-II-OH), r is an integer between 40 and 50.-   Embodiment 88. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein in the PEG lipid of Formula    (PL-II-OH), r is 45.-   Embodiment 89. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein in the PEG lipid of Formula    (PL-II-OH), R⁵ is C₁₇ alkyl.-   Embodiment 90. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PL-II):

wherein r ^(PEG) is an integer between 1 and 100.

-   Embodiment 91. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PEG-1):

-   Embodiment 92. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    Formula (PL-III):

or a salt or isomer thereof, wherein S^(PL1) is an integer between 1 and100.

-   Embodiment 93. The empty LNP or loaded LNP of any one of the    preceding embodiments, wherein the PEG lipid is a compound of    following formula:

-   Embodiment 94. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the phospholipid is DSPC    and the structural lipid is cholesterol.-   Embodiment 95. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the structural lipid is    cholesterol and the PEG lipid is PEG_(2k)-DMG.-   Embodiment 96. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the structural lipid is    cholesterol and the PEG lipid is PEG-1.-   Embodiment 97. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the phospholipid is DSPC    and the PEG lipid is PEG_(2k)-DMG.-   Embodiment 98. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the phospholipid is DSPC    and the PEG lipid is PEG-1.-   Embodiment 99. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the phospholipid is DSPC,    the structural lipid is cholesterol, and the PEG lipid is    PEG_(2k)-DMG.-   Embodiment 100. An empty lipid nanoparticle (empty LNP) comprising a    compound of any one of the preceding embodiments, a phospholipid, a    structural lipid, and a PEG lipid, wherein the phospholipid is DSPC,    the structural lipid is cholesterol, and the PEG lipid is PEG-1.-   Embodiment 101. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the phospholipid is    DSPC and the structural lipid is cholesterol, and one or more    therapeutic and/or prophylactic agents.-   Embodiment 102. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the structural lipid is    cholesterol and the PEG lipid is PEG_(2k)-DMG, and one or more    therapeutic and/or prophylactic agents.-   Embodiment 103. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the structural lipid is    cholesterol and the PEG lipid is PEG-1, and one or more therapeutic    and/or prophylactic agents.-   Embodiment 104. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the phospholipid is    DSPC and the PEG lipid is PEG_(2k)-DMG, and one or more therapeutic    and/or prophylactic agents.-   Embodiment 105. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the phospholipid is    DSPC and the PEG lipid is PEG-1, and one or more therapeutic and/or    prophylactic agents.-   Embodiment 106. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the phospholipid is    DSPC, the structural lipid is cholesterol, and the PEG lipid is    PEG_(2k)-DMG, and one or more therapeutic and/or prophylactic    agents.-   Embodiment 107. A loaded lipid nanoparticle (loaded LNP) comprising    a compound of any one of the preceding embodiments, a phospholipid,    a structural lipid, and a PEG lipid, wherein the phospholipid is    DSPC, the structural lipid is cholesterol, and the PEG lipid is    PEG-1, and one or more therapeutic and/or prophylactic agents.-   Embodiment 108. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising DSPC in an amount from about 0% to    about 20%.-   Embodiment 109. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising cholesterol in an amount from    about 30% to about 50%.-   Embodiment 110. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising PEG_(2k)-DMG in an amount from    about 0% to about 5%.-   Embodiment 111. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising PEG-1 in an amount from about 0%    to about 5%.-   Embodiment 112. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising about 40 mol % to about 60 mol %    of the compound of any one of the preceding embodiments, about 0 mol    % to about 20 mol % DSPC, about 30 mol % to about 50 mol %    cholesterol, and about 0 mol % to about 5 mol % PEG_(2k)-DMG.-   Embodiment 113. The empty LNP or loaded LNP of any one of the    preceding embodiments, comprising about 40 mol % to about 60 mol %    of the compound of any one of the preceding embodiments, about 0 mol    % to about 20 mol % DSPC, about 30 mol % to about 50 mol %    cholesterol, and about 0 mol % to about 5 mol % PEG-1.-   Embodiment 114. The loaded LNP of any one of the preceding    embodiments the encapsulation efficiency of the therapeutic and/or    prophylactic agent is between 80% and 100%.-   Embodiment 115. The loaded LNP of any one of the preceding    embodiments, wherein the wt/wt ratio of the lipid component to the    mRNA is from about 10:1 to about 60:1.-   Embodiment 116. The loaded LNP of any one of the preceding    embodiments, wherein the wt/wt ratio of the lipid component to the    mRNA is about 20:1.-   Embodiment 117. The loaded LNP of any one of the preceding    embodiments, wherein the N:P ratio is from about 5:1 to about 8:1.-   Embodiment 118. A pharmaceutical composition comprising the loaded    LNP of any one of the preceding embodiments and a pharmaceutically    acceptable carrier.-   Embodiment 119. The pharmaceutical composition of any one of the    preceding embodiments, further comprising a cryoprotectant, a    buffer, or a combination thereof.-   Embodiment 120. The pharmaceutical composition of any one of the    preceding embodiments, wherein the cryoprotectant comprises sucrose.-   Embodiment 121. The pharmaceutical composition of any one of the    preceding embodiments, wherein the cryoprotectant comprises sodium    acetate.-   Embodiment 122. The pharmaceutical composition of any one of the    preceding embodiments, wherein the cryoprotectant comprises sucrose    and sodium acetate.-   Embodiment 123. The pharmaceutical composition of any one of the    preceding embodiments, wherein the buffer is selected from the group    consisting of an acetate buffer, a citrate buffer, a phosphate    buffer, and a tris buffer.-   Embodiment 124. A method of delivering a therapeutic and/or    prophylactic agent to a cell within a subject, the method comprising    administering to the subject the loaded LNP of any one of the    preceding embodiments.-   Embodiment 125. A method of specifically delivering a therapeutic    and/or prophylactic agent to an organ of a subject, the method    comprising administering to the subject the loaded LNP of any one of    the preceding embodiments.-   Embodiment 126. A method for the enhanced delivery of a therapeutic    and/or prophylactic to a target tissue of a subject, the method    comprising administering to the subject the loaded LNP of any one of    the preceding embodiments.-   Embodiment 127. A method of producing a polypeptide of interest in a    cell within a subject, the method comprising administering to the    subject the loaded LNP of any one of the preceding embodiments.-   Embodiment 128. A method of treating a disease or disorder in a    subject in need thereof, the method comprising administering to the    subject a therapeutically effective amount of the loaded LNP of any    one of the preceding embodiments.-   Embodiment 129. Use of a loaded LNP of any one of the preceding    embodiments, in the manufacture of a medicament for delivering a    therapeutic and/or prophylactic agent to a cell within a subject.-   Embodiment 130. Use of a loaded LNP of any one of the preceding    embodiments, in the manufacture of a medicament for specifically    delivering a therapeutic and/or prophylactic agent to an organ of a    subject.-   Embodiment 131. Use of a loaded LNP of any one of the preceding    embodiments, in the manufacture of a medicament for the enhanced    delivery of a therapeutic and/or prophylactic to a target tissue of    a subject.-   Embodiment 132. Use of a loaded LNP of any one of the preceding    embodiments, in the manufacture of a medicament for producing a    polypeptide of interest in a cell within a subject.-   Embodiment 133. Use of a loaded LNP of any one of the preceding    embodiments, in the manufacture of a medicament for treating a    disease or disorder in a subject in need thereof.-   Embodiment 134. A loaded LNP of any one of the preceding    embodiments, for use in delivering a therapeutic and/or prophylactic    agent to a cell within a subject, wherein the delivering comprises    administering a therapeutically effective amount of the loaded LNP    to the subject.-   Embodiment 135. A loaded LNP of any one of the preceding    embodiments, for use in specifically delivering a therapeutic and/or    prophylactic agent to an organ of a subject, wherein the delivering    comprises administering a therapeutically effective amount of the    loaded LNP to the subject.-   Embodiment 136. A loaded LNP of any one of the preceding    embodiments, for use in the enhanced delivery of a therapeutic    and/or prophylactic to a target tissue of a subject, wherein the use    comprises administering to the subject the loaded LNP of any one of    the preceding embodiments.-   Embodiment 137. A loaded LNP of any one of the preceding    embodiments, for use in producing a polypeptide of interest in a    cell within a subject, the use comprises administering to the    subject the loaded LNP of any one of the preceding embodiments.-   Embodiment 138. A loaded LNP of any one of the preceding    embodiments, for use in the treatment of a disease or disorder in a    subject in need thereof, wherein the treatment comprises    administering a therapeutically effective amount of the loaded LNP    to a subject.-   Embodiment 139. A method of delivering a therapeutic and/or    prophylactic agent to a cell within a subject, the method comprising    administering to the subject the pharmaceutical composition of any    one of the preceding embodiments.-   Embodiment 140. A method of specifically delivering a therapeutic    and/or prophylactic agent to an organ of a subject, the method    comprising administering to the subject the pharmaceutical    composition of any one of the preceding embodiments.-   Embodiment 141. A method for the enhanced delivery of a therapeutic    and/or prophylactic to a target tissue of a subject, the method    comprising administering to the subject the pharmaceutical    composition of any one of the preceding embodiments.-   Embodiment 142. A method of producing a polypeptide of interest in a    cell within a subject, the method comprising administering to the    subject the loaded LNP of any one of the preceding embodiments.-   Embodiment 143. A method of treating a disease or disorder in a    subject in need thereof, the method comprising administering to the    subject a therapeutically effective amount of the pharmaceutical    composition of any one of the preceding embodiments.-   Embodiment 144. Use of a pharmaceutical composition of any one of    the preceding embodiments, in the manufacture of a medicament for    delivering a therapeutic and/or prophylactic agent to a cell within    a subject.-   Embodiment 145. Use of a pharmaceutical composition of any one of    the preceding embodiments, in the manufacture of a medicament for    specifically delivering a therapeutic and/or prophylactic agent to    an organ of a subject.-   Embodiment 146. Use of a pharmaceutical composition of any one of    the preceding embodiments, in the manufacture of a medicament for    the enhanced delivery of a therapeutic and/or prophylactic to a    target tissue of a subject, the method comprising administering to    the subject the pharmaceutical composition of any one of the    preceding embodiments.-   Embodiment 147. Use of a pharmaceutical composition of any one of    the preceding embodiments, in the manufacture of a medicament for    producing a polypeptide of interest in a cell within a subject.-   Embodiment 148. Use of a pharmaceutical composition of any one of    the preceding embodiments, in the manufacture of a medicament for    treating a disease or disorder in a subject in need thereof.-   Embodiment 149. A pharmaceutical composition of any one of the    preceding embodiments, for use in delivering a therapeutic and/or    prophylactic agent to a cell within a subject, wherein the    delivering comprises administering a therapeutically effective    amount of the pharmaceutical composition to the subject.-   Embodiment 150. A pharmaceutical composition of any one of the    preceding embodiments, for use in specifically delivering a    therapeutic and/or prophylactic agent to an organ of a subject,    wherein the delivering comprises administering a therapeutically    effective amount of the pharmaceutical composition to the subject.-   Embodiment 151. A pharmaceutical composition of any one of the    preceding embodiments, for use in the enhanced delivery of a    therapeutic and/or prophylactic to a target tissue of a subject,    wherein the use comprises administering to the subject the    pharmaceutical composition of any one of the preceding embodiments.-   Embodiment 152. A pharmaceutical composition of any one of the    preceding embodiments, for use in producing a polypeptide of    interest in a cell within a subject, the use comprises administering    to the subject the pharmaceutical composition of any one of the    preceding embodiments-   Embodiment 153. A pharmaceutical composition of any one of the    preceding embodiments, for use in the treatment of a disease or    disorder in a subject in need thereof, wherein the treatment    comprises administering a therapeutically effective amount of the    pharmaceutical composition to a subject.-   Embodiment 154. The method, use, or loaded LNP or pharmaceutical    composition for use, of any one of the preceding embodiments,    wherein the organ is selected from the group consisting of liver,    kidney, lung, and spleen.-   Embodiment 155. The method, use, or loaded LNP or pharmaceutical    composition for use, of any one of the preceding embodiments,    wherein the target tissue is selected from the group consisting of    liver, kidney, lung, and spleen.-   Embodiment 156. The method or loaded LNP or pharmaceutical    composition for use of any one of the preceding embodiments, wherein    the administering is performed parenterally.-   Embodiment 157. The method or loaded LNP or pharmaceutical    composition for use wherein the administering is performed    intramuscularly, intradermally, subcutaneously, and/or    intravenously.-   Embodiment 158. The use of any one of the preceding claims, wherein    the medicament is for parenteral administration.-   Embodiment 159. The use of any one of the preceding claims, wherein    the medicament is for intramuscular, intradermal, subcutaneous,    and/or intravenous administration.-   Embodiment 160. The method, use, or loaded LNP or pharmaceutical    composition for use, of any one of the preceding embodiments,    wherein the subject is human.

EQUIVALENTS

It is to be understood that while the present disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present disclosure, which is defined by the scope of the appendedclaims. Other aspects, advantages, and alterations are within the scopeof the following claims.

1. A compound of Formula (A):

or its N-oxide, or a salt or isomer thereof, wherein m is selected from5, 6, 7, 8, and 9; R² and R³ are each independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl; R⁴ is selectedfrom —(CH₂)_(n)OH, wherein n is selected from 1, 2, 3, 4, and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R¹⁰is —N(R)₂, wherein each R is independently selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₃ alkenyl, and H; M and M′ are eachindependently selected from —OC(O)O— and —C(O)O—; wherein at least oneof M and M′ is —OC(O)O—; R′^(a) is C₁₋₁₈ alkyl or C₂₋₁₈ alkenyl; and R″is a C₃-C₁₃ alkyl, optionally substituted with OH.
 2. The compound ofclaim 1, wherein R″ is selected from

wherein

denotes a point of attachment with N, and

denotes a point of attachment with M.
 3. The compound of claim 1 or 2,wherein the compound has one of the following structures:


4. The compound of any one of the preceding claims, wherein R′^(a) is:

wherein

denotes a point of attachment; R^(aα), R^(aβ), and R^(aγ) are eachindependently selected from the group consisting of H, C₁₋₁₂ alkyl, andC₂₋₁₂ alkenyl, wherein at least one of R^(aα), R^(aβ), and R^(aγ) isselected from the group consisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; andR′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl.
 5. The compound of claim 4,wherein R^(aβ) and R^(aγ) are each H and R^(aα) is C₁₋₆ alkyl.
 6. Thecompound of claim 4, wherein R^(aα) and R^(aγ) are each H and R^(aβ) isC₁₋₆ alkyl.
 7. The compound of claim 6, wherein R^(aβ) is C₂ alkyl. 8.The compound of claim 4, wherein R^(aα) and R^(aβ) are each H and R^(aγ)is C₁₋₆ alkyl.
 9. The compound of claim 8, wherein R^(aγ) is a C₅ or aC₆ alkyl.
 10. The compound of any one of the preceding claims wherein R′is a C₅ or a C₆ alkyl.
 11. The compound of any one of the precedingclaims, wherein the compound has the following structure:


12. The compound of any one of the preceding claims, wherein R⁴ is—(CH₂)_(n)OH.
 13. The compound of any one of the preceding claims,wherein R⁴ is —(CH₂)₂OH.
 14. The compound of any one of the precedingclaims, wherein R⁴ is


15. The compound of any one of the preceding claims, wherein R⁴ is


16. The compound of any one of the preceding claims, wherein n2 is 2 or4.
 17. A compound selected from:


18. An empty lipid nanoparticle (empty LNP) comprising a compound of anyone of the preceding claims, a phospholipid, a structural lipid, and aPEG lipid.
 19. The empty LNP of any one of the preceding claims,comprising about 40 mol % to about 60 mol % said compound, about 0 mol %to about 20 mol % phospholipid, about 30 mol % to about 50 mol %structural lipid, and about 0 mol % to about 5 mol % PEG lipid.
 20. Theempty LNP of any one of the preceding claims, wherein the phospholipidis selected from the group consisting of:1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and mixtures thereof.
 21. The empty LNP of any one of thepreceding claims, wherein the structural lipid is selected from thegroup consisting of cholesterol, fecosterol, sitosterol, ergosterol,campesterol, stigmasterol, brassicasterol, and mixtures thereof.
 22. Theempty LNP of any one of the preceding claims, wherein the PEG lipid isselected from the group consisting of a PEG-modifiedphosphatidylethanolamine, a PEG-modified phosphatidic acid, aPEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modifieddiacylglycerol, a PEG-modified dialkylyglycerol, and mixtures thereof.23. The empty LNP of any one of the preceding claims, wherein the PEGlipid is selected from PEG_(2k)-DMG and PEG-1:

and mixtures thereof.
 24. A loaded lipid nanoparticle (loaded LNP),which comprises the empty LNP of any one of the preceding claims and oneor more therapeutic and/or prophylactic agents.
 25. The loaded LNP ofany one of the preceding claims, wherein the one or more therapeuticand/or prophylactic agents is a nucleic acid.
 26. The loaded LNP of anyone of the preceding claims, wherein the nucleic acid is an RNA, andwherein the RNA is selected from the group consisting of a shortinterfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a RNAinterference (RNAi) molecule, a microRNA (miRNA), an antagomir, anantisense RNA, a ribozyme, a Dicer-substrate RNA (dsRNA), a smallhairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof. 27.The loaded LNP of any one of the preceding claims, wherein the RNA is anmRNA.
 28. A pharmaceutical composition comprising the loaded LNP of anyone of the preceding claims and a pharmaceutically acceptable carrier.29. A method of delivering a therapeutic and/or prophylactic agent to acell within a subject, the method comprising administering to thesubject the loaded LNP of any one of the preceding claims.
 30. A methodof specifically delivering a therapeutic and/or prophylactic agent to anorgan of a subject, the method comprising administering to the subjectthe loaded LNP of any one of the preceding claims.
 31. A method ofproducing a polypeptide of interest in a cell within a subject, themethod comprising administering to the subject the loaded LNP of any oneof the preceding claims.
 32. A method of treating a disease or disorderin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the loaded LNP of any oneof the preceding claims.